TonB-dependent Transport System Research Articles

Tri-layered core-shell structured deferoxamine magnetic particles promote Microcystis aeruginosa growth

This experiment prepared magnetic composite siderophores (DMPs) with strong magnetism, excellent adsorption capacity, and high specific surface area. Exploring the synergistic effect of magnetic nanoparticles and siderophores on Microcystis aeruginosa growth under iron-deficient condition, by utilizing the characteristics of the three-layer core-shell structure of DMPs. This study elucidated the potential mechanism by which DMPs promote the cyanobacterial growth through physiological indicators and transcriptome analysis. On the experiment's final day, cell density in DMPs treatment group at 2, 4, and 8 mg/L were 1.10, 1.14 and 1.16 times higher than those in the control group (Ct), respectively. Similarly, chlorophyll and photosynthetic efficiency results showed improved algae growth with increasing DMPs dosage. The microcystin content in DMPs experimental groups at low, medium, and high concentration were 0.91, 0.86, and 0.83 times that of Ct, indicating alleviation of iron deficiency stress. Additionally, based on extracellular polymers, intracellular and extracellular siderophores, and visualization techniques, DMPs nanoparticles captured free iron sources in the environment, promoting algae growth by entering algal cells and facilitating the uptake and utilization of free iron ions from the solution. During the experiment, the iron uptake and transport genes (feoA and feoB) were significantly upregulated, whereas the algal siderophore synthesis gene (pchF) and the TonB-dependent transport system gene (TonB_C) were significantly downregulated, suggesting heightened activity in intracellular iron uptake and transport. This indicates an abundance of intracellular iron, eliminating the need for secrete siderophores to overcome iron deficiency. Microcystis aeruginosa increased iron bioavailability by using iron transported through DMPs in the environment while internalizing these DMPs. This study explored the mechanism of this synergistic effect to boost algal growth, and provided new ideas for elucidating the mechanism of cyanobacterial bloom outbreaks as well as the innovative application of biotechnology.

Molecular Mechanism of Iron Transport Systems in Vibrio

The ability to acquire iron from the environment is often an important virulence factor for pathogenic bacteria and Vibrios are no exception to this. Vibrios are reported mainly from marine habitats and most of the species are pathogenic. Among those, the pathogenic vibrios eg. V cholerae, V. parahaemolyticus, V. vulnificus causes foodborne illnesses. Vibrios are capable of producing all different classes of siderophores like hydroxamate (aerobactin), catecholate (vibriobactin, fluvibactin), carboxylate (vibrioferrin), and amphiphilic (amphibactin). Every different species of vibrios are capable of utilizing some endogenous or xenosiderophores. Being Gram-negative bacteria, Vibrios import iron siderophore via TonB dependent transport system and unlike other Gamma proteobacteria these usually possess two or even three partially redundant TonB systems for iron siderophore transport. Other than selected few iron siderophores, most pathogenic Vibrios are known to be able to utilize heme as the sole iron source, while some species are capable of importing free iron from the environment. As per the present knowledge, the spectrum of iron compound transport and utilization in Vibrios is better understood than the siderophore biosynthetic capability of individual species.

Vitamin B12-peptide nucleic acids use the BtuB receptor to pass through the Escherichia coli outer membrane

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B12, which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B12 enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B12-specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B12-PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B12. From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B12 transport system can be hijacked to enable PNA delivery to E. coli cells.

Mechanism of TonB-dependent transport system in Halomonas alkalicola CICC 11012s in response to alkaline stress.

Halomonas alkalicola CICC 11012s can grow at pH 12.5, the highest pH at which the organisms in the genus Halomonas can grow. Genomic analysis reveals that H. alkalicola adapts to alkaline stress using a variety of adaptive strategies; however, the detailed mechanism for its growth at high-alkaline conditions has not been elucidated. Therefore, in this study, the adaptations of H. alkalicola in response to extreme alkaline stress were investigated. A sharp decrease of alkaliphilic tolerance was observed in mutants E. coli ΔEctonB and H. alkalicola ΔHatonB. Expressions of the gene clusters encoding TonB-dependent transport system and iron complex transport system in H. alkalicola grown under extreme alkaline conditions were markedly up-regulated. We then compared the intracellular ionic iron content and iron-chelating ability of mutant strain with those of wild-type strain to understand the influence of TonB-dependent transport system on the alkaline responses. The results indicated that the presence of TonB-dependent transport system increased the alkaline tolerance of H. alkalicola grown at high-alkaline conditions, but had no effects when the strain was grown at neutral pH and low-alkaline conditions. Meanwhile, the presence of this system increased the transport and accumulation of ionic irons to maintain intracellular metabolic homeostasis, which in turn could increase the tolerance of the strain to extreme alkaline conditions. Based on the results, we established a model representing the interactions between TonB-dependent transport system, alkaline tolerance, and intracellular ionic iron that could help deepen the understanding of the alkaline response mechanism of alkaliphilic bacteria.

Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB-dependent transport system.

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated Ton complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB-dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

Extracellular loops of BtuB facilitate transport of vitamin B12 through the outer membrane of E. coli.

Vitamin B12 (or cobalamin) is an enzymatic cofactor essential both for mammals and bacteria. However, cobalamin can be synthesized only by few microorganisms so most bacteria need to take it up from the environment through the TonB-dependent transport system. The first stage of cobalamin import to E. coli cells occurs through the outer-membrane receptor called BtuB. Vitamin B12 binds with high affinity to the extracellular side of the BtuB protein. BtuB forms a β-barrel with inner luminal domain and extracellular loops. To mechanically allow for cobalamin passage, the luminal domain needs to partially unfold with the help of the inner-membrane TonB protein. However, the mechanism of cobalamin permeation is unknown. Using all-atom molecular dynamics, we simulated the transport of cobalamin through the BtuB receptor embedded in an asymmetric and heterogeneous E. coli outer-membrane. To enhance conformational sampling of the BtuB loops, we developed the Gaussian force-simulated annealing method (GF-SA) and coupled it with umbrella sampling. We found that cobalamin needs to rotate in order to permeate through BtuB. We showed that the mobility of BtuB extracellular loops is crucial for cobalamin binding and transport and resembles an induced-fit mechanism. Loop mobility depends not only on the position of cobalamin but also on the extension of luminal domain. We provided atomistic details of cobalamin transport through the BtuB receptor showing the essential role of the mobility of BtuB extracellular loops. A similar TonB-dependent transport system is used also by many other compounds, such as haem and siderophores, and importantly, can be hijacked by natural antibiotics. Our work could have implications for future delivery of antibiotics to bacteria using this transport system.

A QUICK WAY TO DETECT, DISCRIMINATE AND QUANTIFY SIDEROPHORES BY FLUORESCENCE ASSAYS

Iron is vital for bacteria, as it plays a central role in energy production, intermediate metabolism, DNA synthesis, nitrogen fixation etc. to survive in host system, Bacteria primarily secrete siderophores that specifically chelate iron with high affinity and actively transport this ferric siderophore complex inside cell. Siderophore production and ferric siderophore acquisition are frequently associated with microbial infections. Moreover, pathogenic bacteria obtain iron with TonB dependent ferric siderophore transport systems. For example, the outer membrane protein IutA actively transports the iron complex ferric aerobactin (FeAero), and the inner membrane protein TonB provides the energy for this uptake reaction. I created fluorescent sensors that monitor high affinity binding reactions, and used them to detect, discriminate and quantify ferric siderophores, as either isolated iron complexes or in complex mixture of metabolites and other biochemicals. By introducing site‐directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells that bind but do not transport target compound. By cloning, genetically engineering and fluoresceinating ferric siderophore transporters, we created specific sensors for the native, degraded and glucosylated forms of the catecholate ferric enterobactin, for the hydroximates ferric aerobactin, ferrichrome and ferrioxamine B, for the porphyrins hemin and vitamin B12and so on. When employed in spectroscopic analysis, these constructs sensitively detected respective ferric siderophores or target compounds and measured their concentrations in solutions. Sensitive Assays of Biochemical specificity, affinity, and capacity are valuable for both basic research and drug discovery. The sensors, which we created monitored production of siderophores by the pathogens in blood samples, food samples or clinical samples, each of which manifested a particular profile of iron chelator production.

TonB-Dependent Transporters in Sphingomonads: Unraveling Their Distribution and Function in Environmental Adaptation.

TonB-dependent transport system plays a critical role in the transport of nutrients across the energy-deprived outer membrane of Gram-negative bacteria. It contains a specialized outer membrane TonB-dependent transporter (TBDT) and energy generating (ExbB/ExbD) and transducing (TonB) inner membrane multi-protein complex, called TonB complex. Very few TonB complex protein-coding sequences exist in the genomes of Gram-negative bacteria. Interestingly, the TBDT coding alleles are phenomenally high, especially in the genomes of bacteria surviving in complex and stressful environments. Sphingomonads are known to survive in highly polluted environments using rare, recalcitrant, and toxic substances as their sole source of carbon. Naturally, they also contain a huge number of TBDTs in the outer membrane. Out of them, only a few align with the well-characterized TBDTs. The functions of the remaining TBDTs are not known. Predictions made based on genome context and expression pattern suggest their involvement in the transport of xenobiotic compounds across the outer membrane.

Comparative genome analysis reveals niche-specific genome expansion in Acinetobacter baumannii strains.

The nosocomial pathogen Acinetobacter baumannii acquired clinical significance due to the rapid development of its multi-drug resistant (MDR) phenotype. A. baumannii strains have the ability to colonize several ecological niches including soil, water, and animals, including humans. They also survive under extremely harsh environmental conditions thriving on rare and recalcitrant carbon compounds. However, the molecular basis behind such extreme adaptability of A. baumannii is unknown. We have therefore determined the complete genome sequence of A. baumannii DS002, which was isolated from agricultural soils, and compared it with 78 complete genome sequences of A. baumannii strains having complete information on the source of their isolation. Interestingly, the genome of A. baumannii DS002 showed high similarity to the genome of A. baumannii SDF isolated from the body louse. The environmental and clinical strains, which do not share a monophyletic origin, showed the existence of a strain-specific unique gene pool that supports niche-specific survival. The strains isolated from infected samples contained a genetic repertoire with a unique gene pool coding for iron acquisition machinery, particularly those required for the biosynthesis of acinetobactin. Interestingly, these strains also contained genes required for biofilm formation. However, such gene sets were either partially or completely missing in the environmental isolates, which instead harbored genes required for alternate carbon catabolism and a TonB-dependent transport system involved in the acquisition of iron via siderophores or xenosiderophores.

Organophosphate hydrolase interacts with Ton components and is targeted to the membrane only in the presence of the ExbB/ExbD complex.

Our study aims to investigate the physiological role of organophosphate hydrolase (OPH), hitherto known for its involvement in the degradation of organophosphate insecticides and nerve agents in Sphingobiumfuliginis. We find that OPH exists as part of the TonB-dependent Transport system that is involved in nutrient transport across the bacterial outer membrane. OPH interacts physically with the Ton complex components ExbD and TonB. The surface-exposed arginine residues (R91 and R96) of OPH facilitate its interaction with ExbD. OPH is targeted to the inner membrane of Escherichiacoli only when it is co-expressed with either ExbD or the ExbB/ExbD complex. In the absence of ExbD, OPH remains in the cytoplasm. Our findings suggest a role for OPH in outer membrane transport.

A Cluster of Five Genes Essential for the Utilization of Dihydroxamate Xenosiderophores in Synechocystis sp. PCC 6803.

The unicellular freshwater cyanobacterium Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron in the TonB-dependent manner. The fecCDEB1-schT gene cluster encoding a siderophore transport system that is involved in the utilization of FeSK and SAV in Synechocystis sp. PCC 6803 was identified. The gene schT encodes TonB-dependent outer membrane transporter, whereas the remaining four genes encode the ABC-type transporter FecB1CDE formed by the periplasmic binding protein FecB1, the transmembrane permease proteins FecC and FecD, and the ATPase FecE. Inactivation of any of these genes resulted in the inability of cells to utilize FeSK and SAV. Our data strongly suggest that Synechocystis sp. PCC 6803 can readily internalize Fe-siderophores via the classic TonB-dependent transport system.

Transition metals at the host-pathogen interface: how Neisseria exploit human metalloproteins for acquiring iron and zinc.

Transition metals are essential nutrients for all organisms and important players in the host-microbe interaction. During bacterial infection, a tug-of-war between the host and microbe for nutrient metals occurs: the host innate immune system responds to the pathogen by reducing metal availability and the pathogen tries to outmaneuver this response. The outcome of this competition, which involves metal-sequestering host-defense proteins and microbial metal acquisition machinery, is an important determinant for whether infection occurs. One strategy bacterial pathogens employ to overcome metal restriction involves hijacking abundant host metalloproteins. The obligate human pathogens Neisseria meningitidis and N. gonorrhoeae express TonB-dependent transport systems that capture human metalloproteins, extract the bound metal ions, and deliver these nutrients into the bacterial cell. This review highlights structural and mechanistic investigations that provide insights into how Neisseria acquire iron from the Fe(III)-transport protein transferrin (TF), the Fe(III)-chelating host-defense protein lactoferrin (LF), and the oxygen-transport protein hemoglobin (Hb), and obtain zinc from the metal-sequestering antimicrobial protein calprotectin (CP).

TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae.

Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention.

A Novel Sialic Acid Utilization and Uptake System in the Periodontal Pathogen Tannerella forsythia

Tannerella forsythia is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of T. forsythia is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of T. forsythia since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of T. forsythia contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in Escherichia coli. This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an Escherichia coli strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the TF0033-TF0034 genes enabled an E. coli nanR nanC ompR strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes nanO and nanU, respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology in vivo.

Structural Evidence for Iron-free Citrate and Ferric Citrate Binding to the TonB-dependent Outer Membrane Transporter FecA

Escherichia coli possesses a TonB-dependent transport system, which exploits the iron-binding capacity of citrate and its natural abundance. Here, we describe three structures of the outer membrane ferric citrate transporter FecA: unliganded and complexed with iron-free or diferric dicitrate. We show the structural mechanism for discrimination between the iron-free and ferric siderophore: the binding of diferric dicitrate, but not iron-free dicitrate alone, causes major conformational rearrangements in the transporter. The structure of FecA bound with iron-free dicitrate represents the first structure of a TonB-dependent transporter bound with an iron-free siderophore. Binding of diferric dicitrate to FecA results in changes in the orientation of the two citrate ions relative to each other and in their interactions with FecA, compared to the binding of iron-free dicitrate. The changes in ligand binding are accompanied by conformational changes in three areas of FecA: two extracellular loops, one plug domain loop and the periplasmic TonB-box motif. The positional and conformational changes in the siderophore and transporter initiate two independent events: ferric citrate transport into the periplasm and transcription induction of the fecABCDE transport genes. From these data, we propose a two-step ligand recognition event: FecA binds iron-free dicitrate in the non-productive state or first step, followed by siderophore displacement to form the transport-competent, diferric dicitrate-bound state in the second step.

TonB-dependent systems of uropathogenic Escherichia coli: aerobactin and heme transport and TonB are required for virulence in the mouse.

The uropathogenic Escherichia coli strain CFT073 has multiple iron acquisition systems, including heme and siderophore transporters. A tonB mutant derivative of CFT073 failed to use heme as an iron source or to utilize the siderophores enterobactin and aerobactin, indicating that transport of these compounds in CFT073 is TonB dependent. The TonB(-) derivative showed reduced virulence in a mouse model of urinary tract infection. Virulence was restored when the tonB gene was introduced on a plasmid. To determine the importance of the individual TonB-dependent iron transport systems during urinary tract infections, mutants defective in each of the CFT073 high-affinity iron transport systems were constructed and tested in the mouse model. Mouse virulence assays indicated that mutants defective in a single iron transport system were able to infect the kidney when inoculated as a pure culture but were unable to efficiently compete with the wild-type strain in mixed infections. These results indicate a role for TonB-dependent systems in the virulence of uropathogenic E. coli strains.

TonB is required for intracellular growth and virulence of Shigella dysenteriae.

To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.

Mechanism of tonB-dependent transport of KP-736, a 1,5-dihydroxy-4-pyridone-substituted cephalosporin, into Escherichia coli K-12 cells.

The mechanism of transport of KP-736, a novel cephalosporin with a 1,5-dihydroxy-4-pyridone moiety at the C-7 position, into the Escherichia coli K-12 cell was investigated by determining the susceptibilities of iron transport mutants to KP-736. The tonB mutant showed a higher degree of resistance to KP-736, indicating that KP-736 was incorporated into E. coli cells via the tonB-dependent iron transport system. The product of the exbB gene was also necessary for the maximal antibacterial potency of KP-736. Cir-lacking and Fiu-lacking mutants showed a moderate level of resistance to KP-736. However, mutants lacking any one of the proteins FepA, FecA, FhuA, and FhuE did not show any increased resistance to KP-736. Two types of spontaneous mutants (e.g., KT1004 and KT1011) could be isolated from cir and fiu mutants by selection for KP-736 resistance and showed the same level of resistance to KP-736 as a tonB mutant. KT1004 showed tonB phenotypes, resistance to phage phi 80, and loss of FecA, whereas KT1011 did not. KT1011 lost the ability to express both Cir and Fiu proteins. These results indicate that the Cir and Fiu outer membrane proteins are involved specifically in the tonB-dependent transport process of KP-736. Against OmpF- and OmpC-deficient transformants producing various groups of beta-lactamases, KP-736 was more effective than the other cephalosporins tested.

Mode of action of GR69153, a novel catechol-substituted cephalosporin, and its interaction with the tonB-dependent iron transport system

GR69153 is a novel cephalosporin incorporating a catechol-substituted 7-aminothiazolyl-oxime. The antibiotic is actively transported into gram-negative cells via iron-regulated outer membrane proteins regulated by the tonB product. This transport enhances bactericidal activity most significantly at low concentrations, essentially removing the permeability barrier for antibiotic uptake.

Primary structure of colicin M, an inhibitor of murein biosynthesis

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.