RopB transcriptional regulator, also known as RopB/Rgg transcriptional regulator is a

transcriptional regulator In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA ( transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from alt ...

protein that regulates expression of the extracellularly secreted cysteine protease streptococcal pyrogenic exotoxin B (speB or streptopain), which is an important

virulence factor Virulence factors (preferably known as pathogenicity factors or effectors in botany) are cellular structures, molecules and regulatory systems that enable microbial pathogens (bacteria, viruses, fungi, and protozoa) to achieve the following: * c ...

of ''

Streptococcus pyogenes ''Streptococcus pyogenes'' is a species of Gram-positive, aerotolerant bacteria in the genus '' Streptococcus''. These bacteria are extracellular, and made up of non-motile and non-sporing cocci (round cells) that tend to link in chains. They ...

'' and is responsible for the dissemination of a host of infectious diseases including

strep throat Streptococcal pharyngitis, also known as streptococcal sore throat (strep throat), is pharyngitis (an infection of the pharynx, the back of the throat) caused by ''Streptococcus pyogenes'', a Gram-positive bacteria, gram-positive, group A str ...

impetigo Impetigo is a contagious bacterial infection that involves the superficial skin. The most common presentation is yellowish crusts on the face, arms, or legs. Less commonly there may be large blisters which affect the groin or armpits. The les ...

, streptococcal

toxic shock syndrome Toxic shock syndrome (TSS) is a condition caused by Exotoxin, bacterial toxins. Symptoms may include fever, rash, skin peeling, and low blood pressure. There may also be symptoms related to the specific underlying infection such as mastitis, ...

necrotizing fasciitis Necrotizing fasciitis (NF), also known as flesh-eating disease, is an infection that kills the body's soft tissue. It is a serious disease that begins and spreads quickly. Symptoms include red or purple or black skin, swelling, severe pain, fever ...

, and

scarlet fever Scarlet fever, also known as scarlatina, is an infectious disease caused by ''Streptococcus pyogenes'', a Group A streptococcus (GAS). It most commonly affects children between five and 15 years of age. The signs and symptoms include a sore ...

. Functional studies suggest that the ropB multigene

regulon In molecular genetics, a regulon is a group of genes that are gene regulation, regulated as a unit, generally controlled by the same regulatory gene that gene expression, expresses a protein acting as a repressor or activator (genetics), activator ...

is responsible for not only global regulation of virulence but also a wide range of functions from stress response, metabolic function, and two-component signaling. Structural studies implicate ropB's regulatory action being reliant on a complex interaction involving

quorum sensing In biology, quorum sensing or quorum signaling (QS) is the process of cell-to-cell communication that allows bacteria to detect and respond to cell population density by gene regulation, typically as a means of acclimating to environmental disadv ...

with the leaderless peptide signal speB-inducing peptide (SIP); acting in conjunction with a pH sensitive histidine switch.

Discovery

Observations of an extracellularly secreted

glucosyltransferase Glucosyltransferases are a type of glycosyltransferase that enable the transfer of glucose. Examples include: * glycogen synthase * glycogen phosphorylase Glycogen phosphorylase is one of the phosphorylase enzymes (). Glycogen phosphorylase c ...

(gtfG) sequentially proximal to and activated by an rgg gene with

inverted repeat An inverted repeat (or IR) is a single stranded sequence of nucleotides followed downstream by its complementarity (molecular biology), reverse complement. The intervening sequence of nucleotides between the initial sequence and the reverse complem ...

s in the

intergenic region An intergenic region is a stretch of DNA sequences located between genes. Intergenic regions may contain functional elements and junk DNA. Properties and functions Intergenic regions may contain a number of functional DNA sequences such as p ...

of '' Streptococcus gordonii'' served as a basis for studying its homology between ''Streptococcus pyogenes''. It was discovered that ''S. pyogenes'' also shared an rgg/ropB gene located directly next to the subject of its transcriptional regulation, in this case speB

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

, with intergenic inverted repeats. Confirmation of linkage between rgg/ropB and speB secretion activation was achieved by means of ropB insertional disruption which resulted in decreased speB production.

Structure

Gene location

The location of the ropB gene is directly and sequentially proximal to the subject of its transcriptional regulation, speB, which lies downstream of a 941 bp intergenic region between the two. Transcription of the ropB gene seems to necessitate a promoter within a series sequences between 238 and 480 bp and up to 800 bp upstream of the gene itself inside the highly repetitive intergenic region.

Protein binding location

The ropB protein binding location lies adjacent to speB promoter 1 that is also located within the highly repetitive intergenic region, although the ropB gene and the speB gene are transcribed in opposite directions. The -10 and -35 regions of speB promoter 1 have poor consensus; in order to ameliorate this, the ropB aids the

RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that catalyzes the chemical reactions that synthesize RNA from a DNA template. Using the e ...

bondage with the help of a polyU

polypyrimidine tract {{Short description, A pyrimidine-rich sequence involved in pre-messenger RNA maturation The polypyrimidine tract is a region of pre-messenger RNA (mRNA) that promotes the assembly of the spliceosome, the protein complex specialized for carrying ...

inside the

palindromic A palindrome ( /ˈpæl.ɪn.droʊm/) is a word, number, phrase, or other sequence of symbols that reads the same backwards as forwards, such as ''madam'' or '' racecar'', the date " 02/02/2020" and the sentence: "A man, a plan, a canal – Pana ...

inverted repeat region in a fashion uncannily similar to

intrinsic termination Intrinsic, or rho-independent termination, is a process to signal the end of transcription and release the newly constructed RNA molecule. In bacteria such as ''E. coli'', transcription is terminated either by a rho-dependent process or rho-indep ...

in ''

E. coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...

''.

Protein domains

N-Terminal

The

N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

domain consists of

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s 1-56 and is an

amino terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

responsible for DNA-binding and is a key mediator in the linkage between the

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

domain of the opposite dimer. The dimer interface II has its I255

side chain In organic chemistry and biochemistry, a side chain is a substituent, chemical group that is attached to a core part of the molecule called the "main chain" or backbone chain, backbone. The side chain is a hydrocarbon branching element of a mo ...

located in the N-terminal.

C-Terminal

The C-terminal domain, also known as ropB-CTD, is a carboxy terminal ligand-binding domain made of amino acids 56–280. RopB-CTD houses 5 TPR motifs and attaches to the SIP

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

in the innermost part of the SIP

binding pocket In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding si ...

in a sequence-specific manner without induction of

polymerization In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many fo ...

TPR domain

The

tetratricopeptide repeat The tetratricopeptide repeat (TPR) is a structural motif. It consists of a degenerate 34 amino acid tandem repeat identified in a wide variety of proteins. It is found in tandem arrays of 3–16 motifs, which form scaffolds to mediate protein– ...

domain provides the concave surface necessitated for SIP recognition. RopB-CTD houses 5 stacked TPR motifs, each having sets of paired antiparallel helices that aid in the formation of a concave inner pathway and a convex exterior. The base of the recognition site is constructed by

alpha helices An alpha helix (or α-helix) is a sequence of amino acids in a protein that are twisted into a coil (a helix). The alpha helix is the most common structural arrangement in the secondary structure of proteins. It is also the most extreme type of l ...

α6 and α8, while the supporting walls are constructed from helices α2, and α12. The exterior portion of the recognition site is flanked by

asparagine Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...

s N152 and N192, thus providing a ridge of support for the peptide-protein complex.

Dimer interface

The dimer interfaces of ropB are constructed by a union of the α8 - α12 helices of the N-terminal domain and the C-terminal domain. Additionally, there is an Interface I forged from three side chains (C22, Y224, and R226), an Interface II forged from one side chain (I255), and N-terminal domains that are all responsible for dimerizing ropB protein subunits together.

Peptide binding pocket

The SIP peptide binding pocket is the docking station of the eight amino acid leaderless peptide signal, speB-inducing peptide (SIP). The binding pocket is a tripartite construction of the C-terminal's α12 helix which is a capping helix, TPR3's α6 helix that has a

hydrophobic In chemistry, hydrophobicity is the chemical property of a molecule (called a hydrophobe) that is seemingly repelled from a mass of water. In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, thu ...

interplay with SIP sidechains, and TPR 4's α8 helix which electrostatically stabilizes SIP. Variations in pH level altered strength of adherence between SIP and the SIP binding pocket with acidic pH levels between 5.5 and 6.5 enhancing adherence and pH levels between 7 and 9 reducing adherence.

Histidine switch

Though the ropB protein has seven

histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...

s (H12, H81, H93, H144, H265, H266, and H277) structurally present, the ropB histidine switch primarily operates with a single functionally involved histidine (H144) conveniently placed to associate with ropB sidechains (Y176 and E185) that near each other upon the addition of a

hydrogen ion A hydrogen ion is created when a hydrogen atom loses or gains an electron. A positively charged hydrogen ion (or proton) can readily combine with other particles and therefore is only seen isolated when it is in a gaseous state or a nearly particl ...

to H144 in acidic conditions. Only one histidine (H12) is located on the N-domain while the rest lie in the C-terminal domain.

Regulon kinetics

''Streptococcus pyogenes'' has evolved an interwoven complex of gene regulatory mechanisms in the SIP signaling pathway by implanting a pH sensitive histidine switch onto the quorum-sensing ropB protein. During the neutral to basic pH conditions whether synthetically induced or naturally caused by low

population density Population density (in agriculture: Standing stock (disambiguation), standing stock or plant density) is a measurement of population per unit land area. It is mostly applied to humans, but sometimes to other living organisms too. It is a key geog ...

of ''S. pyogenes'', the interaction between the unprotonated functionally involved histidine (H144) with relevant sidechains (Y176, Y182, E185) in the SIP binding pocket domain is impaired and speB protease expression is inhibited. On the other hand, as extracellular pH decreases to be more acidic in cases of high population density, ''S. pyogenes'' has no elaborate pH

homeostatic In biology, homeostasis (British also homoeostasis; ) is the state of steady internal physical and chemical conditions maintained by living systems. This is the condition of optimal functioning for the organism and includes many variables, su ...

capabilities relative to non-lactic bacteria, therefore intracellular

cytosol The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells ( intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...

ic pH levels will more easily resemble extracellular levels. Cytosolic acidification mobilizes the SIP pathway to allow for the SIP-ropB protein complex to form and increasing SIP production. Furthermore, increased cytosolic acidity enhances the maturation of speB

zymogen In biochemistry, a zymogen (), also called a proenzyme (), is an inactive precursor of an enzyme. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the activ ...

(speBz) into mature speB protease (speBm) to dramatically increase its

proteolytic Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Protein degradation is a major regulatory mechanism of gene expression and contributes substantially to shaping mammalian proteomes. Uncatalysed, the hydrolysis o ...

activity and virulence.

Homology

Rgg family

Rgg-like transcriptional regulators can be found in a variety of

gram-positive bacteria In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain ...

. Where ropB regulates speB protease production in ''S. pyogenes'', a roughly equivalent secretory control mechanism can be seen in Rgg's regulation of gtfG glucosyltransferase production in ''S. gordonii'', in the manner in which gadR regulates acid resistance in ''

Lactococcus lactis ''Lactococcus lactis'' is a gram-positive bacterium used extensively in the production of buttermilk and cheese, but has also become famous as the first genetically modified organism to be used alive for the treatment of human disease. ''L. la ...

'', how lasX regulates expression of

lantibiotic Lantibiotics are a class of polycyclic peptide antibiotics that contain the characteristic thioether amino acids lanthionine or methyllanthionine, as well as the unsaturated amino acids dehydroalanine, and 2-aminoisobutyric acid. They belong ...

lactocin S in ''

Lactobacillus sakei ''Latilactobacillus sakei'' is the type species of the genus ''Latilactobacillus'' that was previously classified in the genus ''Lactobacillus''. It is homofermentative; hexoses are metabolized via glycolysis to lactic acid as main metabolite; ...

'', and mutR's regulation of mutacin in '' S. mutans''. Sequentially, these genes are all localized contiguously to their respective subject of regulation and share promoters localized contiguously to inverted repeat regions.

RRNPP family

Characterization of the RRNPP family of quorum-sensing regulators (which stands for proteins Rap, NprR, PrgX, PlcRd) were used in comparisons with ropB to postulate its structural functions. The Rap protein derived from

Bacilli Bacilli is a Taxonomy (biology), taxonomic Class (biology), class of bacteria that includes two orders, Bacillales and Lactobacillales, which contain several well-known pathogens such as ''Bacillus anthracis'' (the cause of anthrax). ''Bacilli'' ...

regulates

sporulation In biology, a spore is a unit of sexual (in fungi) or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plant ...

, the NprR protein in ''

Bacillus thuringiensis ''Bacillus thuringiensis'' (or Bt) is a gram-positive bacteria, gram-positive, soil-dwelling bacterium, the most commonly used biological pesticide worldwide. ''B. thuringiensis'' also occurs naturally in the gut of caterpillars of various types ...

'' regulates necrotrophism, the PrgX protein regulates

conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form *Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change o ...

in ''

Enterococcus faecalis ''Enterococcus faecalis'' – formerly classified as part of the group D '' Streptococcus,'' is a Gram-positive, commensal bacterium naturally inhabiting the gastrointestinal tracts of humans. Like other species in the genus '' Enterococcus'', ' ...

'', and PlcR protein regulates transcription of virulence factors in both ''Bacillis thuringiensis'' and ''

Bacillus cereus ''Bacillus cereus'' is a Gram-positive bacteria, Gram-positive Bacillus, rod-shaped bacterium commonly found in soil, food, and marine sponges. The specific name, ''cereus'', meaning "waxy" in Latin, refers to the appearance of colonies grown o ...

''. Similarities were observed in conserved asparagine residues on the TPR motifs of each of these proteins and in ropB.

Quorum sensing

Quorum sensing In biology, quorum sensing or quorum signaling (QS) is the process of cell-to-cell communication that allows bacteria to detect and respond to cell population density by gene regulation, typically as a means of acclimating to environmental disadv ...

regulates a menagerie of aspects in

Bacillota The Bacillota (synonym Firmicutes) are a phylum of bacteria, most of which have Gram-positive cell wall structure. They have round cells, called cocci (singular coccus), or rod-like forms (bacillus). A few Bacillota, such as '' Megasphaera'', ...

including the production of ropB-like proteins in ''

Streptococcus pneumoniae ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, hemolysis (microbiology), alpha-hemolytic member of the genus ''Streptococcus''. ''S. pneumoniae'' cells are usually found in pairs (diplococci) and do not f ...

'' and ''S. pyogenes''. Similarities in the pH sensitivity of the cell signaling mechanisms were found in

pneumococci ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, alpha-hemolytic member of the genus ''Streptococcus''. ''S. pneumoniae'' cells are usually found in pairs (diplococci) and do not form spores and are non mo ...

, '' S. mutans'', and ''

Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often posi ...

'' as well.

pH sensitive histidine switch

Amongst Rgg-like proteins, it has been observed that the pH sensitive histidine (particularly H144) and interacting amino acids (Y176, Y182, and E185) of ropB of ''Streptococcus pyogenes'' are conserved in ''S. porcinus'', ''S. pseudoporcinus'', '' S. salivarius'', ''L. pentosus'', ''L. aviaries'', '' L. reuteri'', and ''

Enterococcus ''Enterococcus'' is a large genus of lactic acid bacteria of the phylum Bacillota. Enterococci are Gram-positive cocci that often occur in pairs ( diplococci) or short chains, and are difficult to distinguish from streptococci on physical ch ...

'' sp. including ''

E. faecalis ''Enterococcus faecalis'' – formerly classified as part of the group D ''Streptococcus,'' is a Gram-positive, commensal bacterium naturally inhabiting the gastrointestinal tracts of humans. Like other species in the genus ''Enterococcus'', ''E ...

''. Thus, suggesting the usage of a pH sensitive histidine switch complex with gene-regulating effector molecules in a slew of other bacteria ee Also: allosteric regulation">allosteric regulation">ee Also: allosteric regulation

Pathogenesis

RopB regulation speB is a key determinant in the expression of the speB proteinase which is a primary virulence factor and the most abundant extracellular protein in streptococcal secretions. SpeB cleaves host serum proteins that make up the human extracellular matrix and bacterial proteins including other secreted streptococcal proteins. As previously mentioned, it is responsible for the dissemination of a host of infectious diseases including but not limited to pharyngitis,

streptococcal toxic shock syndrome ''Streptococcus'' is a genus of gram-positive spherical bacteria that belongs to the family Streptococcaceae, within the order Lactobacillales (lactic acid bacteria), in the phylum Bacillota. Cell division in streptococci occurs along a single ...