UNIVERSITY OF CINCINNATI April 20 , 2001 I, ____Lisa Louise Anderson______, hereby submit this as part of the requirements for the degree of: _____Doctorate of Philosophy______in:___Chemistry______
It is entitled: _____A Study of Penicillin Binding Proteins in______Mycobacterium tuberculosis______Approved by:
Richard A. Day H. Brian Halsall Allan Pinhas ______
ii
A STUDY OF PENICILLIN BINDING PROTEINS IN MYCOBACTERIUM TUBERCULOSIS
A dissertation submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY (Ph.D.)
in the Department of Chemistry of the College of Arts and Sciences
2001
by
Lisa L. Anderson
B.S., Purdue University, 1991 M.S., University of Cincinnati, 1999
Committee Chair:
Richard A. Day
iii
ABSTRACT
Penicillin-class antibiotics, known as b-lactams, are one of medicine's most valuable
weapons against bacterial disease. These drugs infiltrate susceptible bacteria and interrupt normal
growth via interactions with a class of cell wall-synthesizing enzymes known as penicillin-binding
proteins (PBPs). Some pathogens, however, can escape such a fate.
Beta-lactam antibiotics are not currently prescribed for the treatment of tuberculosis,
although it has now been shown that commercially available lactams can bind to PBPs in
membrane fractions from Mycobacterium tuberculosis (MTB), the causative agent.12 13 This lab
has previously documented that PBPs form native protein complexes within H. influenzae and E.
coli.14 15 If similar PBP complexes could be identified within mycobacteria, it would improve our
understanding of the enzymology of cell wall biosynthesis in these organisms. Mycobacterial
PBPs and the proteins with which they naturally interact are valuable targets for antibiotic
research, especially in light of the emergence of multidrug-resistant tuberculosis (MDRTB) in
certain populations worldwide.
The approach outlined here covalently labels PBPs within intact cells before protein
isolation; the advantage provided by this scheme is the opportunity to cross-link PBPs in their
native protein-protein associations. Cross-linking can take place after interaction with b-lactam
tags, but before disruption of cells. This lab has previous experience exploring the topography of
12 Chambers, H. F., Moreau, D., Yajko, D., Miick, C., Wagner, C., Hackbarth, C., Kocagoz, S., Rosenberg, E., Hadley, W., Nikaido, H. (1995) Antimicrob. Agents and Chemo. 39, 2620-2624. 13 Basu, J., Chattopadhyay, R., Manikuntala, K., Chakrabarti, P. (1992) J. Bacteriol. 174, 4829-4832. 14 Alaedini, A., Day, R. A. (1999) Biochem. Biophys. Res. Commun. 264, 191-195. 15 Bhardwaj, S., Day, R. A. (1998) Techniques in Protein Chemistry VIII, Academic Press, San Diego: 469. iv
bacterial PBPs using cyanogen (ethanedinitrile) as a cross-linking agent.3 4 Cyanogen has been shown to permeate intact cells and to covalently crosslink native PBP complexes.
Dansylated penicillin has been used to covalently label purified serine protease enzymes, though to the best of our knowledge it has not been used to label mycobacterial proteins.16 When accompanied by b-lactamase inhibitors, b-lactams interfere with the growth of mycobacteria in cultures17 18 19 20 and intracellularly within macrophages.21 This project sought to label PBPs and associated enzymes in whole MTB cells with a fluorescent tag, then to isolate these proteins. Both a de novo monocyclic lactam and a commercially available penicillin were dansylated in order to investigate their effectiveness in labeling PBPs within whole mycobacterial cells. We found that mycobacterial PBPs could be fluorescently tagged and cross-linked to associated proteins within intact cells.
16 Fink, A. L., Cartwright, S. J. (1989) Biochem. J. 263, 905-912. 17 Sorg, T., Cynamon, M. (1987) J. Antimicrob. Chemother. 19(1), 59-64. 18 Prabhakaran, K., Harris, E, Randhawa, B, Adams, L, Williams, D., Hastings, R.(1993) Microbios 76(309),251- 61. 19 Chen, C., Yand, M., Lin, J., Lee, Y., Perng, R. (1995) Proc. Nat. Sci. Coun. Repub. China B. 19(2), 80-84. 20 Herbert, D., Paramasivan, C., Venkatesan, P., Kubendiran, G., Prabhakaran, K., Mitchison, D. (1996) Antimicrob. Agents Chemother. 40(10), 2296-9. v
21 Prabhakaran, K., Harris, E., Randhawa, B. (1999) Int. J. Antimicrob. Agents 13(2), 133-5. vi
ACKNOWLEDGMENTS
Many thanks to those who taught me something about chemistry, and who gave me the chance to learn by teaching as well. Endless thanks to my family for giving me so much love and opportunity my whole life.
1
Table of Contents
List of Tables………………………………………………………….………………..3 List of Figures……………………………………………………………….………….4 1. Introduction………………………………………………………………………….6 1.1. Mycobacterial disease………………………………………………….…..6 1.2. Therapeutic regimens…………………………………………………..…..7 1.3. Drug resistance……………………………………………………………..9 1.4. Mycobacterial cell wall…………………………………………………...13 1.5. Permeability barrier…………………………………………………….....16 1.6. Peptidoglycan biosynthesis…………………………………………….....17 1.7. PBP complexes…………………………………………………….……...19 1.8. Discovery within mycobacteria…………………………………….……..22 1.9. Cyanogen as a cross-linking reagent…………………………….………..22 2. Objectives……………………………………………………………….……...…..25 3. Materials and Methods…………………………………………………….……….27 3.1. Microbiological cultures…………………………………….…………....27 3.2. Synthetic methods………………………………………….……………..29 3.3. Methods for detecting PBPs and complexes……………….……………..36 3.4. Analytical methods…………………………………….…………….…....42 4. Results and Discussion…...…………………………………….……………….….46 4.1. Microbiology……………………………………….……………….…….46 4.2. Synthetic products………………………………….………………....…..48 4.2.1. Leuchs anhydride of glycine………………….………….……..48 4.2.2. Monocyclic lactam……………………………….……….….....50 4.2.3. DAPA…………………………………………….………..…....52 4.2.4. Acylated commercial lactams……………………..………...…..54 4.3. PBPs and protein complexes………………………………..………..…...59 4.3.1. Labeling……………………………………..……………..…....59 2
4.3.2. Cell fractionation and protein partition………………..…………63 4.3.3. Electrophoresis and electroblots………………………...……….64 4.3.4. Protein molecular weight estimation…………………….………65 4.3.5. Cyanogen cross-linking………………………………..………...67 4.3.6. Mass spectrometry of peptide digests…..….……………………69 4.3.7. Competitive binding assay…………………………...……….....72 5. Conclusion…………………………………………………………...…………..…74 6. Appendix 1.a. Putative M. tuberculosis PBPs…………..…………..……………...76 Appendix 1.b. Peptide fingerprint matches for 66kDa protein…………………….78 Appendix 1.c. Peptide fingerprint matches for 85kDa protein…………………….85 Appendix 1.d. Peptide fingerprint matches for protein complex…………………..89
3
List of Tables
1. Spontaneous mutation rate in resistance-causing mycobacterial genes…….…...9 2. Functions of penicillin binding proteins in E. coli……………………………..18
4
List of Figures
1. First line of defense chemotherapeutics used against tuberculosis…………..…..8 2. Development of antibiotic resistance……………………………………….…...10 3. Beta-lactam functionality………………………………………………………..12 4. Envelope of mycobacteria……………………………………………………….13 5. Mycolic acids linked to peptidoglycan………………………………….…...…..14 6. Long-chain fatty acids of mycobacteria……………………………………….....15 7. PBP cross-linking reaction……………………………………………………....19 8. Multienzyme peptidoglycan-synthesizing complex model……………………...20 9. Cyanogen reaction…………………………………………………………..…...24 10. Ring closure of carbobenzyloxyglycine…………………………………….…...29 11. Formylation of Leuchs anhydride of glycine………………………………..…..30 12. De novo synthesis of substituted beta-lactam ring…………………….…….…..31 13. Fluorinated dansylated monocyclic lactam probe……………………….…..…..32 14. Dansylation of 6-aminopenicillanic acid…………………….………….…….…33 15. Acetylations of 6-aminpenicillanic acid……………….……………….…….....34 16. Amine groups targeted for acetylation…………………………………..…..…..36 17. Protocol for labeling bacterial proteins……………………………………..…...38 18. Mycobacterial growth curves………………………………………………..…..47 19. NMR spectrum of Leuchs anhydride of glycine……………………………..….48 20. GC-MS of formylated Leuchs anhydride of glycine…………………………....49 21. IR spectrum of formylated Leuchs anhydride of glycine…………………...…..50 22. Bioautography of de novo beta-lactam……………………………………….....51 23. Bioautography of dansyl aminopenicillanic acid………………………………..52 24. IR spectrum of dansyl aminopenicillanic acid…………………………………..53 25. FT-ICR MS of dansyl aminopenicillanic acid…………………………………..54 26. NMR of dansylaminopenicillanic acid………………………………………….55 27. Depiction of bioautography on synthetic products……………………………...56 28. Minimum inhibitory concentrations of acylated lactams……………………….58 5
29. Coomassie-stained PAGE of M. smegmatis proteins……………………….…..61 30. Electroblot of M. smegmatis proteins…………………………………….……..62 31. Control for electrophoresis of dansyl aminopenicillanic acid……………..…..…63 32. Electroblot of solubilized membrane proteins………………………………...…64 33. Timed stages of membrane protein electroblot……………………………….…65 34. M. tuberculosis proteins labeled with DAPA……………………………………66 35. Labeled cyanogen complexes shown by PAGE…………………………….……67 36. Detail of cyanogen complexes shown by PAGE…...………………………….…68 37. Competitive binding of mycobacterial proteins………………………………..…73
6
1. INTRODUCTION
1.1 Mycobacterial Disease
Tuberculosis is a contagious pulmonary disease caused by the rod-shaped bacterium
Mycobacterium tuberculosis (MTB). Closely related organisms of this genus cause leprosy,
Mycobacterium avium Complex (MAC) infection, and mycobacterial disease in animal
populations. Mycobacteria are classified as Gram-positive organisms, but they differ from other bacterial species in their unusual cell wall sugars and lipids, as well as in high (60-70%) genomic
G + C content.22
Streptomycin, an inhibitor of bacterial protein synthesis and the first successful anti-
tubercular drug, was introduced in 1948.23 Although this advance heralded a decline in the prevalence of tuberculosis in the United States, many parts of the world did not benefit to the extent that wealthier populations did. Even now, fifty years after the first clinical successes against this disease, tuberculosis kills between 2 and 3 million people worldwide each year--the largest
number of deaths attributable to a single organism. 24 Tuberculosis control is an international
burden, borne more heavily by lesser-developed countries, but shared by Europe and the U.S.. In
the 1980s, a resurgence of tuberculosis in certain American populations emphasized the need for
wealthier countries to invest more resources in clinical research and surveillance of the threat
posed by MTB. Better management of this public health problem has become even more critical
with the spread of mycobacterial strains that are resistant to the few first-line antibiotics available
to treat tuberculosis.
22 Cole, S., et al. (1998) Nature 393(6685) 537-44. 23 Blanchard, J. S. (1996) Annu. Rev. Biochem. 65, 215-239. 7
1.2 Therapeutic Regimens
The two most important tuberculosis-fighting drugs are isoniazide (INH) and rifampicin
(RMP). Isoniazide (isonicotinic acid hydrazide), which acts on the enzyme used by tubercle
bacilli in elongation reactions during mycolic acid synthesis, has been a mainstay of tuberculosis
treatment since the 1950s; its structural analog ethionamide has also proven to be clinically useful
against mycobacterial infection. Pyrazinamide, the pyrazine analog of nicotinamide, is hydrolyzed
in the liver to pyrazinoic acid, and then can act on phagocytosed mycobacteria. Rifampicin
inhibits bacterial RNA polymerase. A fourth antitubercular drug, ethambutol, is a synthetic
compound thought to target synthesis of the arabinogalactan found in mycobacterial cell walls
(Fig.1).25
Although these agents are relatively non-toxic at therapeutic serum levels, their use is complicated by the growth patterns of mycobacteria in vivo. Besides actively multiplying in the lungs and body tissues, MTB bacteria reside in latent condition within the macrophages of the human immune system. Mitchison distinguishes four distinct populations of mycobacteria within infections: actively metabolizing cells, semi-dormant cells within acidic host environments, semi- dormant cells in non-acidic milieu, and dormant cells.26 These populations differ in their susceptibility to particular antibiotics. INH, for example, is highly active against multiplying bacilli, yet it is not successful at completely wiping out tuberculosis infection due to the survival of persistent semi-dormant bacilli. The standard clinical solution is to administer INH in combination with a "sterilizing" drug--one known to destroy slowly metabolizing populations of mycobacteria. Pyrazinamide, and sometimes rifampicin, are used widely for this purpose.16
24 WHO Global TB Programme (1994-1997), Anti-Tuberculosis Drug Resistance in the World, Geneva. 25 Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C., Yolken, R. H., (1995) Manual of Clinical Microbiol., ASM Press, Washington, D. C. 8
Figure 1. First line of defense chemotherapeutic agents used against tuberculosis.
1.3 Drug Resistance
Another factor confounding the clinical management of tuberculosis is resistance to chemotherapeutics. Mycobacteria have not yet been seen to transmit resistance genes horizontally
26 Mitchison, D. A. (1992) J. Antimic. Agents and Chemo. 29, 477-493. 9
by plasmid exchange, as many other Gram-positive organisms do, but they do experience
spontaneous genetic mutations that result in resistance to chemotherapeutics. The frequencies of
resistance-generating events (spontaneous “mutation rates”) have been established for common
tuberculosis drugs (Table 1).27 Because statistics inform us that one bacillus in 108 will acquire a
natural resistance to INH, for instance, we know that treatment with INH alone can encourage the
growth of a resistant population by eliminating the competition of susceptible mycobacteria.
Drugs that have high "early bactericidal activity" (EBA), however, tend to disfavor the emergence
of resistance by quickly destroying large numbers of exponentially multiplying cells.9 Cases of
infection by bacteria that are resistant to both RMP and INH are classified as "multi-drug
resistant-tuberculosis", or MDRTB, and pose a serious threat to tuberculosis management from a
public health standpoint.
Table 1. Spontaneous mutation rates in resistance-causing mycobacterial genes.
27 Grange, J. M. (1988) Mycobacteria and Human Disease, Edward Arnold Ltd, London, 153-154. 10
Figure 2. Development of antibiotic-resistant tuberculosis.
11
The chance of a single bacillus randomly acquiring genetic resistance to two drugs is the
product of the two individual probabilities, and is thus a highly unlikely event. Clinical
mismanagement or patient noncompliance with medication, however, greatly increases the
likelihood of MDRTB development. In this way, the natural evolution of resistance can be
artificially amplified by antibiotic treatment. When such clinically acquired infection is transmitted
to another individual, it becomes a case of "primary resistance"(Fig. 2).14 The hospital setting is
an especially high-risk environment for transmission of this type of resistant infection.
Because of the limited number of drugs effective in fighting tuberculosis, resistance
phenomena are even more of a problem than with other types of infections. â-lactam antibiotics,
which are medicine's most important class of antimicrobials, are not currently used to cure
tuberculosis. Each compound within this class possesses a four-membered cyclic amide, and this functionality acts as a suicide substrate on cell wall-synthesizing enzymes (Fig. 3).15 While
existing antimycobacterials act on enzymes performing lipid metabolism, penicillin and related
antibiotics interfere with peptidoglycan synthesis.
Mycobacteria have a potent combination of defenses against b-lactams. Two conditions
appear to hinder the clinical effectiveness of b-lactams on tuberculosis: reduced permeability of
the tightly-packed lipid in mycobacterial cell walls, and the presence of b-lactamases, bacterial
enzymes which efficiently hydrolyze lactams before they reach their therapeutic targets.2 28 29 30
Lactams used clinically are hydrophilic molecules; a carboxylic acid group at a critical position in each structure seems to be an important feature for the drugs to be recognized by their target enzymes. The mycobacterial envelope, though technically Gram positive, features a layer of lipid
28 Höltje, J., Said, I. (1983) in: The Target of Penicillin, Walter de Gruyter & Co., New York, 439-444. 29 Romeis, T., Höltje, J. (1994) J. Biol. Chem. 269, 21603-21607. 12
which resembles the outer membrane of Gram-negative bacteria (Fig. 4). The structure of this layer effectively limits, though does not completely prevent, the permeation of hydrophilic molecules into the cell.
Figure 3. The four-membered cyclic amide ring of â-lactam antibiotics.
Once a lactam does cross the cell wall, it encounters a second effective obstacle. â- lactamase enzymes, which exist in a wide array of bacterial species including mycobacteria, hydrolyze b-lactam molecules with remarkable efficiency. MTB possess strong b-lactamase activity capable of destroying both penicillins and cephalosporins. The class A b-lactamase in M.
30 Fattorini, L., Orefici, G., Jin, H. S., Scarcaci, G., Amicosante, G., Franceschini, N., Chopra, I. (1992) Antimic. 13
tuberculosis has been localized to the envelope of these cells. Bush has classified this enzyme as a
group 2b b-lactamase.31 In recent years, researchers have tested the b-lactamase inhibitors
sulbactam and clavulanic acid in combination with ampicillin or amoxycillin, and have observed
significant antimycobacterial activity.32
Figure 4. The mycobacterial envelope, consisting of a thick lipid layer outside the cell wall peptidoglycan.
1.4 Mycobacterial Cell Wall
The mycobacterial cell wall is notable for its complexity and unique composition, which
includes a layer of long-chain fatty acids (mycolic acids) linked via carbohydrate to peptidoglycan,
the standard bacterial cell wall polymer (Fig. 5).33 A number of glycolipids, such as trehalose
mycolates, associate non-covalently with the exterior of the cell wall. These lipids cling to the
Agents and Chem. 36(5), 1068-1072. 31 Bush, K. (1997) Antimic. Agents and Chemo. 41(5), 1182-1185. 32 Utrup, L. J., Moore, T. D., Actor, P., Poupard, J. A. (1995) Antimic. Agents and Chemo. 39(7), 1454-1457. 33 Brennan, P. J., Nikaido, H. (1995) Annu. Rev. Biochem. 64, 29-63.
14
thick, well-packed layer of mycolic acids, which spans approximately 12 nanometers. Within the mycolic acid chains, one or two cyclopropyl groups and cis double bonds may
Figure 5. Mycolic acids are esterified to complex carbohydrates in the outer mycobacterial cell envelope. 15
introduce kinks, but for the most part, these long carbon chains align in a tight side-by-side
conformation perpendicular to the cell wall plane. This hydrophobic structure resembles an outer
membrane; its densely packed arrangement allows little fluidity within the layer, especially in
regions along the extremities of the mycolic acids, which tend not to be interrupted by kink-
inducing groups. Mycolic acids in mycobacteria are typically composed of a saturated 24-carbon
a-branch, and a hydroxylated 60-carbon meromycolate moiety (Fig. 6). Species of mycobacteria differ in the specific structure of meromycolate; differences such as trans versus cis double bond
configuration are expected to influence the packing and permeability of the lipid layer.23
Figure 6. Mycobacteria differ from other bacteria in the exceptional length of the fatty acids found in their cell envelopes. Sites of unsaturation affect packing of meromycolate strands.
Mycolic acids are anchored to inner layers of the cell wall via ester linkages.
Arabinogalactan, a sugar unique to mycobacteria, connects each fatty acid chain to the inner 16
glycan polymer known as peptidoglycan, or murein.23 34 Elucidation of the enzymology of sugar
biosynthesis in mycobacteria has only just begun. Because no other bacteria are known to
produce the rhamnose and arabinose sugars that mycobacteria incorporate into their envelope, the
proteins involved in the synthesis of these sugars are of great interest; one could hope that drugs
designed to target such uncommon enzymes may be highly specific in their action.
1.5 Permeability Barrier
Just as gram-negative bacteria possess channel-forming membrane proteins, or porins, in order to allow the exchange of hydrophilic molecules with their environment, so too do mycobacteria. A single M. chelonae porin spans a diameter of 2 nm.35 The flux allowed by these pores, however, belies the large size of each porin. The performance of porins in bacterial cells is affected not only by pore size, but also by channel depth and the number of channels available. In mycobacterial cells, porins span the lipid layer of approximately 12 nm; in addition they only sparsely populate these cells.
The passage of small hydrophilic molecules (such as b-lactams) into mycobacteria via porins is a very inefficient route of entry. Jarlier and Nikaido measured the permeability coefficients for passage of various cephalosporins and small hydrophilic molecules through the cell envelope of M. chelonei, and found them to be exceedingly low, on the order of 10-8 cm/s. 36
Comparable measurements on Escherichia coli and Pseudomonas aeruginosa were at least ten- fold higher.36 Chambers et al. found that the rates of penetration of â-lactam antibiotics through the outer cell wall of M. tuberculosis H37Ra were approximately one hundred-fold less than rates for E. coli.1 These findings indicated that the mycobacterial permeability barrier “was shown
34 Chatterjee, D. (1997) Curr. Opin. Chem. Biol. 1(4), 579-588. 35 Trias, J., Jarlier, V., Benz, R. (1992) Science 258,1479-1481. 36 Jarlier, V., Nikaido, H. (1990) J. Bacteriol. 172 (1), 1418-1423. 17
to reduce drastically the stream of drug molecules entering the cell, allowing the rather low level
of b-lactamase to decrease radically the concentration of the drug at the target.”36
Some drugs may cross the mycobacterial cell wall in a temperature-dependent manner (D.
Cross, R. Day, unpublished). This suggests that diffusion through the lipid core itself is the major
mode of permeation for these compounds. Also, it has been shown that the more lipophilic
molecules within a certain class of compounds achieve more rapid entry than less lipophilic
compounds of that same class.23 Preliminary high-throughput screening studies performed in this
lab have suggested that fluorination of monocyclic lactams that are b-lactamase-stable37 may yield
active antimycobacterial agents (Cross, D. A., Day, R. A., unpublished). Although the lipid layer
of mycobacterial cell walls is notably low in fluidity, these observations may suggest that diffusion
through it is possibly the most efficient means for drugs to reach their targets.
1.6 Peptidoglycan Biosynthesis
Those enzymes involved in peptidoglycan synthesis serve as good antibiotic targets, since
eukaryotic cells do not manufacture this biopolymer. â-lactams are directed at penicillin-binding proteins (PBPs), a set of enzymes exhibiting various activities necessary for building peptidoglycan and restructuring it during cell division (Table 2).38 The natural function of PBPs is to catalyze peptidase reactions among the pentapeptides linked to the glycan chains of peptidoglycan (Fig. 7).39 Each PBP has serine peptidase activity wherein a serine residue located in its active site acts as a nucleophile. Transpeptidases and D,D-carboxypeptidases attack the carbonyl of a terminal alanyl-alanine peptide bond, and in turn facilitate attack at the same carbon
37 Ahluwalia, R., Day, R., Nauss, J. (1995) Biochem. Biophys. Res. Commun. 206, 577-583. 38 Tipper, D. (1979) Rev. Inf. Dis. 1(1), 39-54. 39 Mathews, C., van Holde, K., Ahern, K., Biochemistry, 3rd Edition (2000) Addison Wesley Longman, Inc., San Francisco. 18
by an amino nucleophile of a peptide acceptor, or by water. Transpeptidases form cross-links by catalyzing the condensation between peptides attached to separate glycan strands. In contrast,
D,D-carboxypeptidases catalyze hydrolytic release of the terminal alanine without transpeptidation. Endopeptidases bind existing cross-linkages, and catalyze their hydrolysis.40
This function would be important during growth and cell wall reshaping.
Table 2. The chemical activities of known E. coli PBPs.
Penicillins and other b-lactams appear to be structurally analogous to the transition state that precedes the peptide bond in the reaction described above. PBPs can recognize and attack b- lactams as they would their natural substrates, yet they cannot rapidly release product from such
40 Tipper, D. (2000) in Antibiotic Inhibitors of Bacterial Cell Wall Biosynthesis Pergamon Press, NY. 19
reactions. Once an active-site serine esterifies the lactam carbonyl carbon, it remains covalently
modified for a deleterious span of time.
Figure 7. PBPs catalyze the formation of peptide cross-links between peptidoglycan strands.
1.7 PBP Complexes
The final assembly of peptidoglycan precursors into the stress-bearing bacterial envelope
takes place on the exterior of the plasma membrane, at the interface with the peptidoglycan.
Because the cell wall must resist high pressures and retain the bacterial shape, the synthesis and
interweaving of new material into existing cell wall structures must be a highly controlled process.
Discussions on the mechanism of murein turnover acknowledge the need for systematic cross-
linking of nascent glucosamine-muramic acid chains to peptidoglycan, while old cross-links are 20
being broken to allow the fitting in of the new strand.41 42 This process must be organized such
that the integrity of the envelope is maintained against several atmospheres of osmotic pressure.
Coordination in time and space of several different enzymatic activities—those of murein
synthases and hydrolases, for example--seems essential. Intramolecular protein interactions seem a
logical means of controlling cooperation. Höltje’s growth model proposes a multienzyme murein-
synthesizing complex which he compares to the holoenzyme that replicates DNA while traveling
along its substrate in a well-orchestrated unit (Fig. 8).32 Nanninga also suggests that interaction
between PBP3 and morphogene proteins such as FtsZ may play a role in cell division.43
Figure 8. The model for a complex of cell-wall synthesizing enzymes operating as a unit to remodel and expand the cross-linked mesh of bacterial peptidoglycan. Some PBPs that have already been characterized have dual functionalities. E. coli PBP1a,
for example, has transglycosylation as well as a D,D-transpeptidase activities.44 Moreover, this
lab has documented evidence of complexation among PBPs and other proteins related to cell
division and morphology.3 It makes biological sense that these functionally related and
41 Höltje, J. (1992) in Bacterial Growth and Lysis, Ed. M. A. de Pedro Plenum Press, NY. 42 Höltje, J., (1998) Microbiol. and Molec. Biol. Rev. 62 (1), 181-203. 43 Nanninga, N. (1998) Microbiol. And Molec. Biol. Rev. 62 (1), 110-129. 21
interdependent enzymes would associate together through specific protein-protein interactions.
The nature of these putative complexes has been the subject of much speculation. Said and Höltje
used the cross-linking reagent dithiobis (succinimidylpropionate) on bacterial membranes to
establish certain of the spatial relationships between PBPs in E. coli . They were able to
demonstrate cross-links among three PBPs, as well as other unidentified proteins, which were
hypothesized to operate in a native complex.18 Later, Höltje and Romeis used affinity
chromatography to show affinity between a soluble transglycosylase and each of two PBPs from
E. coli.19 A 1995 publication described attempts to investigate the putative multi-component PBP
complex in E. coli. In summary, however, it was acknowledged that "if a protein complex of
PBP1a/1b, PBP3 and PBP5 exists then interaction between its constituent proteins is not
detectable by this system."45 They commented that a variety of cross-linking reagents would
benefit future studies. Indeed, in recent years, this lab has successfully applied the cross-linking
reagent cyanogen (ethanedinitrile) to detect native protein-protein interactions in a variety of
systems, including E. coli.3,4
In a basic sense, the study of "protein sociobiology," or the "ability to determine where
and when protein partnerships form in the living cell is of fundamental importance to
understanding biological processes."46 If a PBP complex from mycobacterial species could be
isolated and characterized, new proteins that play a role in cell wall biosynthesis, even perhaps
novel drug targets, may be identified. For this reason, detecting native PBP-protein interactions
would not only be biologically interesting, but would also have medical import.
44 Goffin, C., Ghuysen, J. (1998) Microbiol. Mol. Biol. Rev. 62(4), 1079-1093. 45 Harris, F., Chatfield, L., Phoenix, D. A. (1995) Biochem. Soc. Transactions 562S 23. 46 Day, R. N. (1998) Nature Biotech 16, 514-15. 22
1.8 Discovery within Mycobacteria
Penicillin-binding proteins have indeed been detected and isolated from mycobacteria.
Basu et al. used ampicillin affinity chromatography to purify five PBPs from M. smegmatis cell
extracts. Once labeled with radioactive penicillin, these proteins were characterized by denaturing
polyacrylamide gel electrophoresis as having apparent molecular weights of 94, 67, and 56-46
kDa.2
Chambers et al. also used affinity chromatography, in this case to recover fractions of
MTB cell lysate enriched in PBPs. In addition, these researchers labeled PBPs within mycobacterial membranes with radioactive penicillin. Four labeled PBPs with apparent sizes of
94, 82, 52, and 35 kDa were detected by radioautography of SDS-PAGE gels. Sulbactam, an
inhibitor of b-lactamase, protects the radiolabeled lactam in membrane preparations, but also
binds competitively to the PBPs themselves.1 Despite that the existence of PBPs in MTB has been
documented, these proteins have yet to be fully characterized.
1.9 Cyanogen as a Cross-linking Reagent
Salt bridges are typical features at native protein-protein interfaces. The electrostatic attraction between a positive and negative charge separated by 2.7 angstroms does not contribute as much to the energetics of association as an equivalent hydrophobic contact would, but the specificity of such one-on-one pairings is expected to contribute greatly to guiding the correct inter- and intra-molecular arrangement of protein side chains. Theoretical calculations by Sindelar et al. reinforce suggestions that "these essentially destabilizing electrostatic interactions may play 23
a role in defining specificity, the uniqueness of the lowest free-energy state”47 at protein
interfaces. Ground-state degeneracy may be reduced by the need to compensate buried charges
via salt bridges and hydrogen bonds. Whatever their subtle structural purposes, salt bridges that
occur between proteins in native complexes are valuable targets for the biochemist interested in
investigating these complexes. Many reagents are known to transform salt bridges into permanent
covalent links, thereby cementing natural interactions so that they may be studied in the
laboratory. Bifunctional cross-linking reagents, however, insert atoms into intrinsic structures,
and so are likely to alter spacing between linked groups. Moreover, artifacts result when these
reagents link groups that may not naturally be in stable interaction.
Cyanogen, a small, non-polar cross-linker that is soluble in water as well as organics, can
easily penetrate cells. Not only is it a simple task to introduce this gaseous reagent into the
cellular milieu without perturbing the environment, but it is also a specific method of linking only
stably paired ions. Cyanogen drives condensations between salt bridges buried within proteins or
stable protein interfaces; it has not been shown to form any other inter-molecular covalent bonds
between charged groups (Fig. 9).48 49 The mechanism of cyanogen action appears to resemble
that seen in mono-functional carbodiimide cross-linking reactions. Cyanogen provides us with
the critical tool needed to accomplish our ultimate goal: to covalently cross-link PBP complexes within mycobacterial cells before disrupting the native intact cell envelope.
47 Sindelar,C. V., Hendsch, Z. S., Tidor, S. (1998) Pro. Sci. 7, 1898-1914. 48 Day, R. A., Tharp, R. L., Madis, M. E., Wallace, J. A., Silanee, A., Hurt, P., Mastuserio, N. (1990) Peptide Res. 3, 169-175. 49 Day, R. A., Gooden, W. E., Hignite, A. (1995) Techniques in Protein Chemistry VI , Academic Press, San Diego, 435-442. 24
Figure 9. Cyanogen has been shown to drive covalent bond formation between stable salt bridges.
2. OBJECTIVES
25
The preliminary phases of research detailed here used rapidly growing mycobacterial strains as models to establish basic experimental techniques and as indicators to evaluate anti- bacterial activity of synthetic products.
M. fortuitum grew rapidly and copiously under our lab conditions; in addition, this organism has been used in MIC experiments as an indicator for antibacterial activity of various compounds against MTB.50 This organism was therefore used for biological assays of compounds synthesized in our work. M. aurum, which also grew relatively quickly, was chosen as a model organism for labeling experiments due to its similarity to MTB in lipid composition.
Of our repertoire of mycobacterial test organisms, M. aurum most closely matched MTB in percentage fraction of trans double bonds at the proximal position of the a-mycolate; these organisms have been documented as having <20% and <10%, respectively, of these double bonds in the trans configuration.23 As noted earlier, this arrangement is expected to influence packing, and therefore permeability, of the lipid layer of the mycobacterial envelope.
The research objective centered on proteins isolated from M. tuberculosis, a slowly growing strain of mycobacteria; cultures of attenuated MTB were therefore used for labeling experiments performed at later stages of the project. We intended to develop a protocol for treating whole MTB cells with fluorescent â-lactam, and then associating fluorescently-tagged protein isolates with records in publicly available genomic sequences which have been identified by homology as penicillin binding proteins.
Finally, after labeling PBPs in whole MTB cells, we aimed to cross-link these labeled proteins in native enzyme complexes by treating the intact cells with cyanogen. Because PBPs are 26
expected to cooperate closely with other proteins in peptidoglycan biosynthesis, this procedure could make it possible to recover a collection of interdependent enzymes while making use of a single well-defined affinity label. The ultimate purpose of this work is to demonstrate that cyanogen can be used to cross-link PBPs in native protein complexes within intact mycobacterial cells. The accomplishment of this goal will provide a foundation for later investigations into the enzymology of mycobacterial cell wall biosynthesis.
50 Chung, G., Aktar, Z., Jackson, S., Duncan, K. (1995) Antimic. Agents and Chemother. 39(10), 2235-2238. 27
3. MATERIALS AND METHODS
3.1 Microbiological Cultures
3.1.1 Source of Mycobacteria
Freeze-dried Mycobacterium tuberculosis cells, strain H37Ra, were obtained from
American Type Culture Collection (ATCC Number: 25177). As an attenuated strain, this culture
has been assigned a Centers for Disease Control Biosafety Level 2 rating on a scale from 1 to 4,
with Level 4 being the most hazardous classification. Biosafety Level 2 agents are considered to
pose a moderate potential danger to humans, and may produce disease when not contained by
good laboratory techniques.
Stock cultures of M. aurum and M. fortuitum were available in the lab. Fresh subcultures were started in broth medium then used to grow colonies on Lowenstein-Jensen egg-based solid media slants and Middlebrook 7H10/OADC agar plates for storage at 4°C. New stock cultures grown on solid media were checked for identity and quality, then dedicated for use by a single investigator.
3.1.2 Growth Conditions
All cultures were incubated at 37°C. MTB cultures were incubated at 5% CO2.
Freeze-dried MTB cells were rehydrated and revived to active growth by incubation in
Middlebrook 7H9 broth, which had been aseptically supplemented with ADC enrichment after
sterilization by autoclaving. Cells were subcultured once in liquid medium and incubated for 20
days, then pelleted by centrifugation and re-suspended in double-strength skim milk that had been
autoclaved at 113-115°C for 20 minutes on three consecutive days to destroy contaminating 28
spores. Aliquots of 0.2 mL of cell suspension were quick-frozen in a dry ice-ethanol bath and
stored at -80°C. Pure stock cultures of M. aurum, M. fortuitum and M. tuberculosis were
maintained on solid agar plates (Middlebrook 7H10 medium, supplemented with OADC
enrichment after sterilization by autoclaving) at 4°C. Working stock cultures in 7H9/ADC broth
were kept at 4°C, then used to inoculate fresh broth subcultures as needed for individual
experiments.
3.1.3 Quantitation of Cells
Light absorbance of MTB in 7H9/ADC broth was correlated to the number of colony forming units (CFU), or viable cells, per milliliter of culture. A Bausch & Lomb Spectronic 20 was calibrated with a MacFarland No. 0.5 barium sulfate turbidity standard (0.05 mL 1% BaCl2,
9.95 mL 1% H2SO4) was prepared and measured. A nine-day MTB culture in 7H9/ADC was
diluted to match the standard absorbance, and serial dilutions of this adjusted broth culture in
sterile saline (0.9% NaCl) were plated on 7H10/OADC medium and incubated until colonies were
visible. Colony counts were used to calculate the number of CFU in the spectrophotometrically
adjusted broth. The cultures used in antibiotic assays were diluted to the desired inoculum size
using this reference standard.
3.1.4 Purity of Cultures
Steps were taken to ensure the purity of experimental cultures, such as spot-checking
supplemented media for sterility by incubating it overnight before inoculating it with
mycobacteria. Periodically, heat-fixed smears of stock cultures were treated with Ziehl-Neelsen
carbolfuschin stain and examined for non-acid-fast contaminants. A control smear of M. 29
smegmatis obtained from cultures maintained in the University of Cincinnati undergraduate microbiology lab was stained to serve as an acid-fast positive control for the initial examination.
Mycobacteria, bacilli from 0.3-0.6 x 0.5-0.6 microns in size, are distinctive in their susceptibility to staining with phenol-based dye, and then to resist decolorization with acidic alcohol.
Homogeneity and distinct colony morphology on agar plates also served as indications of culture purity.
Growth rates of bacterial strains were monitored spectrophotometrically. Tween 80 was added to broth used for growth experiments (final concentration 0.02% Tween) in order to avoid clumping, a noted characteristic of MTB. Measurements of light absorbance at l=550 or 650 nm were taken using the Spec20 instrument.
Figure 10. Cyclization of carbobenzyloxyglycine by intramolecular nucleophilic attack yields Leuchs anhydride of glycine (GLA).
30
3.2 Synthetic Methods
3.2.1 Monocyclic Lactam by De Novo Synthesis
Upon conversion to its acid chloride derivative, carbobenzyloxyglycine undergoes a very
favorable intramolecular nucleophilic attack that results in cyclization of the molecule. Quantities
of approximately 250 millimoles of carbobenzyloxyglycine were refluxed in a 2.8-fold molar
excess of thionyl chloride until by-product HCl gas being routed through a sodium carbonate trap had dissipated; reaction reached completion in roughly 20 minutes. Product was isolated by vacuum filtration and washed free of benzyl chloride with 100-200 mL diethyl ether. Peptide
chemists know the cyclic N-carboxy anhydride obtained as glycine-based Leuch’s anhydride
(GLA)51 (Fig. 10).
Pure GLA was formylated at the alpha carbon (Fig. 11). The reaction was carried out by
refluxing at 110 C in toluene, using sodium hydride as a base and t-butyl formate or n-butyl formate as the acylating agent. After 4-5 hours of refluxing, hydrogen evolution slowed drastically, and the reaction was cooled and rotary evaporated. The crude mixture was carried on without purification; for some syntheses or analyses crude Na-FLAG was protonated by treating with acidified Dowex 50W-X12 400-mesh cation exchange resin in methanol.
Figure 11. Butyl formate acylation of GLA under reflux with strong base.
51 Jones, J. (1992) Amino Acid and Peptide Synthesis, Oxford University Press. 31
Epsilon-dansyl-L-lysine (38 mg) and Na-FLAG (250 mg) were stirred from 12-48 hours with ethanol, methanol, or trifluoroethanol (2 mL). The reaction was brought to a pH of ~6 with pyridinium acetate (3 mL acetic acid, 3 mL pyridine), then diluted one hour later with 3 mL deionized H2O. According to the synthetic scheme developed by Day, this drop in pH after the
Na-FLAG has reacted with an alcohol and an amine, promotes an intramolecular attack by the amine on a ring carbonyl. The result is decarboxylation, and contraction of the ring to a four- membered lactam (Fig. 12).27 After 15 minutes, an acylating reagent, either acetic anhydride or a trifluoroacetylating agent, was added and stirred for an additional 45 minutes to 1 hour. Solution was diluted with ethanol (20 mL), then rotary evaporated at ~40°C. The crude mixture was lyophilized and the active agent was extracted with methyl isobutyl ketone (Fig. 13).
Figure 12. Scheme for de novo synthesis of monocyclic â-lactams.
32
Figure 13. The synthetic target: a fluorinated, dansylated â-lactam for use as a fluorescent mycobacterial PBP label.
3.2.2 Dansylated Penicillanic Acid
Dansyl chloride was condensed with 6-aminopenicillanic acid according to the method of
Cartwright and Fink (Fig. 14).5 Adjustments in reaction concentration were made to improve the yield of the dansylation reaction; the absolute concentration of dansyl chloride was kept greater than or equal to 5 mM. Aminopenicillanic acid (0.35 mmol) was dissolved in 50/50 (v/v) 3% aqueous sodium bicarbonate/acetone (45 mL). Dansyl chloride (0.37 mmol) was dissolved in 7 mL acetone, and then added to the reaction mixture. The reaction was stirred at room temperature, and the pH was maintained between 8 and 9 with 3 M NaOH. Consumption of aminopenicillanic acid was monitored by thin layer chromatography (TLC), using ninhydrin to detect the free amine. When necessary, excess dansyl chloride was added to consume unreacted amine. Reaction time was from three to five hours.
Unreacted dansyl chloride was removed by washing the reaction mixture three times with diethyl ether. The pH was adjusted to 2.5 with 3 M HCl; the solution was then saturated with
NaCl and extracted with ethyl acetate. The first extract fluoresced yellow-green under ultraviolet light. Additional extractions appeared bluish upon UV irradiation-possibly due to the presence of 33
dansyl hydroxide. The extracts were examined by TLC, then dried over magnesium sulfate and
evaporated under vacuum.
Figure 14. Dansylation of 6-aminopenicillanic acid to form DAPA.
3.2.3 Fluorinated Derivatives of Commercial Lactams
â-lactam antibiotic precursors were targeted for modification at primary amine groups.
The goal was to find reaction conditions appropriate for acetylating control compounds as well as for trifluoroacetylating test compounds of interest (Fig. 15, 16). Three different approaches were
attempted to achieve acylation at the desired position.
Jones has detailed well-known coupling reactions which use dicyclohexylcarbodiimide
(DCC) in the presence of a nucleophile such as N-hydroxysuccinimide or 1-hydroxybenzotriazole
to form acylating agents.41 In this scheme, once the carboxylate of interest reacts with DCC to form a highly reactive O-acylisourea, the nucleophilic additive attacks to form an acylating agent
which is still reactive to aminolysis, but is less prone to interfering side-reactions such as intramolecular acyl transfer. 34
Figure 15. Acetylation and trifluoroacetylation of 6-aminopenicillanic acid.
Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide were combined in equimolar amounts in dry distilled tetrahydrofuran at 20°C (1 mmol each in 7 mL THF). With reactants stirring, 60 ml (1 mmol) glacial acetic acid was added. The reaction flask was purged with nitrogen and stirred for 15 minutes. Then, 1 mmol of aminopenicillanic acid was added. The reaction was stirred and cooled for 4-5 hours, filtered, then dried to an oil.
Buckwell et al. acetylated penicillanic acid using a "mixed anhydride" method in aqueous solution.52 To a dioxane (3 mL), acetone (1 mL) and triethyl amine (1.5 mmol) stirring over ice,
1.5 mmol of acetyl chloride was added. Methyl chloroformate (1.5 mmol) was added dropwise, and stirred for 45 minutes at temperatures from 3 to 10° C. A cooled solution of 6-APA (1 mmol) and triethyl amine (1 mmol) was prepared in 3 mL of water. This solution was added to 35
the mixed anhydride solution and stirred for one hour at 0-5°C. Sodium bicarbonate (3%, 7 mL) was added, and the reaction was washed with diethyl ether. The aqueous layer was acidified with dilute HCl, and then extracted twice with ether. The extracts were washed with ice water, then dried and evaporated.
Brain et al. used trisubstituted acetic acids and 6-APA to synthesize "trisubstituted methyl penicillins."53 Their conditions were adapted for use with acylating agents of interest. Acetyl chloride (0.3 mmol) in 1.8 mL acetone was added over 15-30 minutes to a stirred solution of 6-
APA (0.3 mmol) in 3% sodium bicarbonate (2.5 mL) and acetone (0.75 mL). After 4-5 hours stirring at room temperature, reaction was washed with ether, acidified, and then extracted with ether.
Acylations were carried out in organic solvent per Hoover et al. 54 Triethyl amine (0.26 mmol) and 6-APA, ampicillin, or 7-ACA (0.1 mmol) were dissolved in anhydrous chloroform
(250 ìL) and chilled to 3°C. Acetyl chloride (0.14 mmol) in chloroform (250 ìL) was added slowly over 1 hour. The reaction mixture was stirred at room temperature for 3 hours, then cooled and acidified with dilute H2SO4. Chloroform extracts were re-extracted with H2O at basic pH. These aqueous fractions were acidified and extracted with diethyl ether.
This protocol was also applied to trifluoroacetylations. According to Schallenberger and
Calvin, S-ethyl trifluoroacetate is a useful reagent for trifluoroacetylating peptides; therefore, this compound was chosen for our experiments on lactams.55
52 Buckwell, S., Page, M. (1988) J. Chem. Soc. Perkin Trans II, 1809-1813. 53 Brain, E., Doyle, F., Hardy, A. (1962) J. Chem. Soc., 1445. 54 Hoover, J., Chow, A., Stedman, N., et al. (1964) J. Med Chem 7(3), 245. 55 Schallenberg, E., Calvin, M. (1955) JOC 77, 2779-2783.
36
Figure 16. The amine groups of ampicillin and 7-aminocephalosporanic were targets for acylation.
3.3 Experimental Methods for Detecting PBPs and Complexes
3.3.1 Labeling Proteins in Whole Cells
Log-phase or stationary-phase mycobacterial cells were harvested by centrifugation at
8,000-10,000 x g for 20 minutes. Pellets were suspended in phosphate-buffered saline (PBS,
10mM phosphate, 150mM NaCl, pH 7.4) and pelleted again by centrifugation at 10,000 x g for
10 minutes. Wet weight was determined for each pellet, then cells were treated with the
fluorescent lactam and/or other compounds of interest dissolved in PBS. As a guideline for
timing and relative amount of treatment compounds, we used the knowledge that "for clinical
use, sulbactam is combined with ampicillin...in a 1:2 ratio," and that the half-life of both antibiotics
in serum is approximately one hour.15 Control pellets were left untreated. Samples were either
incubated at 37°C for a span of time from 30 minutes to several hours, or rotary shaken at room temperature overnight. After incubation, cells were pelleted and washed repeatedly with PBS
(Fig. 17). 37
For competition assays, unlabeled membrane pellets were pre-exposed to penicillin in
combination with â-lactamase inhibitor. Pellets were first washed with 10 mM sodium phosphate
buffer. To the test pellets, a solution of sulbactam (10 mg/mL) and penicillin G (200 mg/mL) in 50
mM sodium phosphate was added at approximately 1 ml per milligram of membrane. Control
pellets were treated with a similar buffer solution containing only 10 mg/mL sulbactam. All pellets
were incubated for 20 minutes at 37°C, then treated with 200 mg/mL DAPA for 30 minutes at
37°C.
3.3.2 Subcellular Fractionation
Cell pellets were suspended in lysis buffer (50mM Tris-HCl). Suspended mycobacteria were lysed either by three passes through a French pressure mini-cell, cooled on ice in between runs, or with five 3-minute periods of sonication on ice using a Model W140 Heat Systems
Ultrasonics probe. Unbroken cells were pelleted by centrifugation for 10 minutes at 10,000 x g.
Supernatant was transferred to clean, thick-walled 13-mL polycarbonate ultracentrifuge tubes and centrifuged in a Beckman 40Ti rotor at 35,000 rpm (100,000 x g) for 1 to 3 hours. Under adequate vacuum within the instrument chamber, rotor temperature was maintained at 4°C throughout the run. After cytosolic fractions were decanted, membrane pellets were washed with lysis buffer and pelleted again. 38
Figure 17. Protocol for labeling and preliminary isolation of mycobacterial PBPs.
3.3.3 Partitioning
For some experiments, membrane proteins were partitioned between an aqueous and a detergent phase per Bordier56 in order to separate peripheral from integral membrane proteins.
56 Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607. 39
Mehotra et al. adapted this method to separate membrane proteins from M. fortuitum.57 Aliquots
of 2% Triton X-114 in Tris-buffered saline (TBS, 10mM Tris, 150mM NaCl, pH 7.4) were added
to membrane pellets on ice, and incubated at 0-4°C with gentle intermittent mixing for one hour.
Upon warming to 37°C and brief centrifugation, two distinct phases formed and were separated.
Detergent and aqueous fractions were washed by mixing and chilling with TBS or Triton X-114,
respectively, then repeating the separation step. Protein from pooled detergent fractions was
precipitated with five volumes of chilled acetone; proteins were precipitated from TBS using
ammonium sulfate or concentrated by ultrafiltration in 10,000 MW cutoff Centricon devices.
3.3.4 Solubilization
For most experiments, protein samples were prepared for electrophoresis under standard conditions according to Protein Methods.58 Membrane proteins, however, are prone to
aggregation upon unfolding. Unfolding protein at high temperatures, then allowing solutions to
cool and potentially aggregate may enhance this effect. Thus the standard procedure for
solubilizing proteins prior to gel electrophoresis, which entails boiling protein samples in buffer
containing 0.4% sodium dodecyl sulfate, was modified for a series of later experiments in hopes
of improving resolution of protein bands on gels and blots. These protein samples were handled
according to the protocol of von Jagow and Schägger, in which a fourfold concentration of
sample buffer was prepared at 12% SDS, then diluted and incubated with membrane pellets at
57 Mehotra, J., Bisht, D., Tiwari, V., Sinha, S. (1995) Clin. Exp. Immunol. 102, 626-634. 58 Bollag, D. M., Rozycki, M. D., Edelstein, S. J., Eds. (1996) Protein Methods, Second Edition, Wiley-Liss, NY. 40
40°C for 30 minutes.59 The buffer also contained 6% mercaptoethanol, 1% bromophenol blue,
50% glycerol, and 150 mM Tris.
3.3.5 Gel Electrophoresis
Both 10% and 12% polyacrylamide 10-well gels were run on a Novex minigel apparatus.
All 10% gels were prepared manually in the lab, while 12% gels were purchased from Fisher
Scientific. Sample aliquots were typically 30 ml in volume, and were prepared to contain an estimated protein load of 20-100 mg. Current was started at a potential of 100 volts, then stepped up to 200 V once samples had entered the stacking gel. Run time was approximately 50 minutes.
Gels were either fixed immediately and stained with silver per Shevchenko et al.,60 or by
Coomassie Blue, or electroblotted and then stained with Coomassie to allow detection of residual proteins. Various molecular weight standard proteins were either weighed and combined, or purchased as premixed MW markers.
3.3.6 Electroblotting
Immediately after electrophoresis, polyacrylamide gels were removed from their cassettes
and equilibrated briefly in Tris-glycine transfer buffer. Polyvinylidinedifluoride (PVDF)
membranes were wetted in methanol, rinsed with deionized water, and then equilibrated in
transfer buffer. Membranes and gels were assembled in a Biorad Trans-Blot electrotransfer tank filled with chilled transfer buffer, and a constant current of 300 mAmps (60-80 V) was applied.
During blotting, the tank was chilled with ice to minimize the effects of heat transfer.
59 Von Jagow, G., Schägger, H., Eds. (1994) A Practical Guide to Membrane Protein Purification, Academic Press, San Diego. 60 Shevchenko, A., Wilm, M., Vorm, O., Mann, M. (1996) Anal. Chem. 68, 850-858. 41
In order to optimize transfer time, a PVDF membrane was cut into five strips, each the
width of two sample lanes. A distinctive notch pattern was cut into the lower edge of each strip
for identification. An electrophoresis gel containing proteins from sulbactam/dansyl-APA -treated
M. aurum cells was arranged on the strips in the electroblot cassette and constant current of 300
mAmps was applied. At intervals from 30 minutes to 3 hours, the current was stopped briefly and
individual PVDF strips were removed from the cassette.
3.3.7 Cross-linking of Native Protein Complexes
Cyanogen (C2N2, ethanedinitrile) was obtained commercially from Apogee Technology of
Wayne, New Jersey. After treatment with test compounds, but before sonication, washed mycobacterial cells were suspended in TBS and sealed in centrifuge tubes with rubber septa, or in glass 5-mL conical reaction vials with Teflon septa. A 5-mL gas-tight syringe was used to draw air from the headspace over the cell suspension, then to inject 5 mL of cyanogen gas into the tube.
Samples were gently agitated for 5 to 20 minutes; solution changed color from off-white to light golden. Cyanogen was purged from the headspace of treated samples with nitrogen gas.
3.3.8 Proteolytic Digests
In-gel digestion of fluorescent bands was carried out per Shevchenko et al.50 Gel pieces
were placed in Eppendorf vials and covered with acetonitrile to dehydrate. Solvent was removed,
then replaced with 10 mM dithiothreitol in 10 mM ammonium bicarbonate in order to reduce
disulfide bonds in the protein samples. After one hour of incubation at 56°C, gel fragments were
decanted, then covered with 55 mM iodoacetic acid in 100 mM NH4HCO3 buffer and protected
42
from light for 45 minutes. Gel pieces were decanted and washed with 100 mM NH4HCO3 for 10
minutes. Fragments were dehydrated with acetonitrile, rehydrated with 100 mM NH4HCO3, then
reshrunk again with acetonitrile. Liquid was completely removed by decanting then centrifuging
samples under vacuum.
Ice-cold digestion buffer containing 5 mM CaCl2 and 12.5 ng/ml trypsin or chymotrypsin
in 50 mM NH4HCO3 was added in order to swell gel pieces. Samples were iced for 45 minutes.
Supernatant was then removed and replaced with ~10 ml of the same buffer. Digestion was allowed to proceed overnight at 37°C.
Peptides were extracted at room temperature with three changes of 5% formic acid in 50% acetonitrile at 20-minute intervals. A microbalance was used to approximate mass of material recovered for mass spectrometry experiments.
3.4 Analytical Methods
3.4.1 Minimum Inhibitory Concentration Assay
MIC values were determined by broth microdilution method in Mueller-Hinton cation-
adjusted medium. M. fortuitum was used as a rapidly growing indicator organism; it has been
shown to exhibit peak macroscopic growth after 72 hours of incubation in this type of assay. 61
Inoculations contained 105 cells per milliliter of broth, according to spectroscopic standardizations.
3.4.2 Bioautography
The components of crude synthetic mixtures that possessed antibiotic activity were 43
detected by thin-layer chromatography combined with microbiological assay.62 Silica-coated
TLC plates were spotted with samples and developed in 3:1:1 n-butyl alcohol: acetic acid: water.
Residual solvent interfered with bioassay unless plates were thoroughly dried, either under
vacuum or overnight at ambient conditions. Dried plates were applied to Mueller-Hinton agar
petri plates containing either Bacillus subtilis spores throughout or log-phase M. fortuitum or M.
tuberculosis cells diluted in a thin overlay of a 45%-strength recipe of Mueller-Hinton agar.
Compounds were allowed to diffuse into agar for at least 15 minutes at 4°C. TLC plates were removed with tweezers and plates incubated at 37°C until a visible lawn of bacterial growth developed.
3.4.3 Modified Lowry Procedure
The Lowry Assay for protein quantitation was modified by Markwell et al. to
accommodate more SDS needed for solubilization of membrane samples.63 Mycobacterial
membrane pellets were subjected to this Modified Lowry Procedure in order to estimate the
amount of membrane protein which was recovered from cultures. Ovalbumin or bovine serum
albumin was used as a control, and measurements of light absorbance at l=660nm were compared
to the standard plot produced by Markwell.53
3.4.4 High-Performance Liquid Chromatography
61 Wallace, R. J., Nash, D. R., Steele, L. C., Steingrube, V. (1986) J. Clin. Microbiol. 24(6), 976-981. 62 Stahl, E., Ed. (1969) Thin-Layer Chromatography: A Laboratory Handbook, George Allen & Unwin Ltd, London.
63 Markwell, M., Haas, S., Bieber, L., Tolbert, N. (1978) Analyt. Biochem. 87, 206-210. 44
HPLC was performed on a Perkin Elmer 250 Binary LC Pump system equipped with a
Perkin Elmer 235 diode array ultraviolet absorbance detector. Reversed-phase columns were
obtained from Rainin (C18, 300 angstrom particle size).
Protein samples tend to adsorb irreversibly to the hydrophobic column packing under
chromatographic conditions, and so 100 microliter injections of trifluoroethanol were used
between runs. TFE is known to induce helix formation in random-coil proteins, and thus appears
to be capable of dislodging proteins which are adsorbed on column packing.64
3.4.5 Infrared Spectroscopy
Infrared (IR) spectra were obtained on a Perkin Elmer spectrometer. Unless noted
otherwise, samples were deposited as a thin film on a calcium fluoride plate for measurement.
3.4.6 NMR Spectroscopy
Nuclear Magnetic Resonance (NMR) spectra were acquired on a Brücker 250 MHz
instrument.
3.4.7 Mass Spectrometry
Small organic molecules were analyzed using a gas chromatograph-mass spectrometer
(GC-MS), or fourier transform ion cyclotron resonance spectrometer (FT-ICR) as noted in discussion sections. Peptide digest analyses were performed on two MicroMass spectrometers equipped with time-of-flight mass analyzers: an ESI Q-TOF which uses electrospray ionization,
64 Bhardwaj, S., Day, R. (1995) LC-GC 17(4), 354-56.
45
and a MALDI-TOF which uses matrix-assisted laser desorption ionization. The digests were also tested on a MALDI-TOF instrument by Brücker.
46
4. RESULTS AND DISCUSSION
4.1 Microbiology
4.1.1 Growth Curves of Mycobacterial Strains
The lag phase of bacterial growth decreases in length for repeatedly subcultured cell
populations. For this reason, growth curves were measured on cultures that had been inoculated
from stocks kept at 4°C rather than cells revived directly from a freeze-dried state. Penicillin-
binding proteins are expected to be most abundant in rapidly dividing bacterial cells, and
documenting growth phases is essential for timing the most efficient cell harvest. Actively dividing
cells are numerous in broth culture late in the exponential phase of growth known as “log phase,”
just before bacterial populations stabilize into a stationary phase of growth.
All mycobacterial strains are relatively slowly-growing bacteria; optimal harvest times were determined on the order of days. M. fortuitum cultures were considered to be in late log phase growth on day 3 after subculture, while optimal harvest time for M. aurum cultures was day
5. M. tuberculosis grew much more slowly; growth days 10, 11, and 12 were targeted for harvesting MTB cultures (Fig. 18).
4.1.2 Colony-forming units in Broth Culture
The MacFarland No. 0.5 barium sulfate optical standard had an A625 of 0.089. MTB
broth culture adjusted to match this optical standard was calculated to contain approximately
1.1x107 to 3x107 viable bacilli per milliliter. Murray noted that a 0.5 MacFarland standard should
8 15 have an A625 of 0.08-0.10, and should correspond to approximately 10 CFU per milliliter. Our 47
data agreed with this approximation, and allowed us to execute MIC assays with standardized
inoculum sizes.
Figure 18. Growth of mycobacterial species. Blue diamonds: M. tuberculosis; pink squares: M. aurum; yellow triangles: M. fortuitum.
4.1.3 Macroscopic Growth and Acid-fast Stains of Mycobacteria
M. fortuitum and MTB colonies were cream in color and irregularly shaped. MTB appeared in crumbly and dry-looking colonies on solid media, and in small (0.5 mm) clumps in
broth culture, while M. fortuitum grew in smooth, flat, moist-looking colonies. M. aurum cells
were a distinctive melon-orange color; membranes and crude membrane proteins isolated from
these cells retained this color, presumably due to lipid-bound chromophores.
Microscopic examination of samples from M. tuberculosis cultures after Ziehl-Neelson staining revealed bright pink bacilli. In the event that culture quality was questionable due to differences in appearance or growth patterns, the cultures were either destroyed or examined for 48
staining properties before use. Non-acid-fast contaminants, when observed, appeared as blue
(counterstained) cocci or coccobacilli.
4.2 Evaluation of Synthetic Products
4.2.1 Leuchs Anhydride of Glycine
The product of the cyclization reaction, the first step in de novo lactam synthesis, was an
opaque golden-colored fine crystalline solid. No further purification of ether-washed product was necessary. The GLA dissolved in deuterated dimethyl sulfoxide for NMR spectroscopy; and the
1H NMR spectrum obtained was simple (4.2 (2H, s), 8.8 (1H, brs)), in agreement with predicted structure (Fig. 19). Crystals of product abruptly released gas, presumably by decarboxylation, upon heating to 90-100°C or treatment with acidic alcohol. Yield for this reaction was typically greater than 90%.
Figure 19. 1H NMR spectrum of GLA. Chemical shifts: 4.2 (2H, s), 8.8 (1H, s). 49
Figure 20. GC-mass spectrum of protonated FLAG.
Formylated Leuchs Anhydride of Glycine
The sodium salt of the enolate form of the desired product (Na-FLAG) was a minor
component of the crude light tan powder recovered. After taking into account the presence of the
side product sodium butoxide, and glycine, a major competing product, gross yield calculations
indicate that the content of Na-FLAG in the solid recovered could be no higher than 30%.
The protonated form of the desired product was recovered in crude form as a clear yellow
oil after cation exchange with the Dowex 50W-X12 400-mesh cation exchange resin. GC-Mass spectrometry experiments with this crude product showed a component having a molecular weight and simple fragmentation consistent with the proposed structure (Fig. 20). The oil exhibited a strong, broad IR band at 1713 cm-1 (5.8 mm), consistent with an acid anhydride 50
carbonyl (Fig.21). In addition, samples of the oil were separated by RPLC on a C18 column.
Fractions absorbing at 220 nm were isolated and examined by IR spectroscopy; the peak observed
at 1679 cm-1 (5.9 mm) is consistent with the absorbance of a b-keto aldehyde in enol form or
absorbance of a urethane group.
Figure 21. The IR spectrum of protonated FLAG (thin film on calcium fluoride plate) showed a strong absorbance at 1713 cm-1 (5.8 mm), consistent with an acid anhydride carbonyl.
4.2.2 Monocyclic Lactam
The reaction was monitored by thin layer chromatography (TLC) on silica plates eluted with 3:1:1 n-butanol:acetic acid:water; a series of minor fluorescent components were observed after dansyl-lysine was allowed to react with Na-FLAG (the dansyl group emits strong yellow- green fluorescence under ultraviolet light). Addition of pyridinium acetate buffer to lower 51
reaction pH obscured the fluorescence of all but the least polar of these components. Methylation of this mixture with diazomethane prepared from N-nitroso-N-methyl urea yielded an mixture of three well-resolved fluorescent components (Rf=0.57, 0.71, 0.85) which were significantly less polar than pyridinium (Rf=0.25, broad) by TLC analysis. The crude mixture was dissolved in 80% acetonitrile for separation by RPLC. Fractions which absorbed at 320 nm were collected and pooled based on TLC analysis, then lyophilized. MALDI mass spectrometry experiments on isolated fractions provided no evidence of expected structure.
Unmethylated crude product subjected to TLC, then bioautographic development revealed a fluorescent component with Rf=0.42; upon bioautographic development on B. subtilis, a clear kill zone which appeared to correspond to this component has been observed, but could not be consistently reproduced (Fig. 22).
Figure 22. Bioautography on a lawn of B. subtilis indicated antimicrobial activity of a reaction mixture component with Rf= 0.42, which also fluoresced under ultraviolet light.
52
4.2.3 Dansyl-Aminopenicillanic Acid (DAPA)
The fluorescent product of the 6-APA dansylation reaction had an Rf=0.68 on silica when eluted with 3:1:1 n-butanol:acetic acid:water. Bioautographic development indicated that this component possessed antibiotic activity. The controls, dansyl chloride (Rf=0.39, 0.95) and 6-
APA (Rf=0.25), did not exhibit significant biological activity (Fig. 23). When the dansylation reaction was driven forward with a high concentration of dansyl chloride, the extracted product showed little or no impurity by TLC. The dry bright yellow solid was used for labeling experiments with no further purification.
Figure 23. The product of 6-aminopenicillanic acid dansylation reactions showed antimicrobial activity upon B. subtilis bioautography. Dotted lines indicate spots on the TLC plate which fluoresced under ultraviolet light. 53
DAPA possessed a strong IR band at 1784 cm-1 (5.6 mm), consistent with the expected
absorbance for a carbonyl stretching in a fused b-lactam. The band at 1147 cm-1 (8.7 mm) is consistent with the presence of a sulfonamide bond linking the penicillanic acid amine group to the dansyl chromophore (Fig. 24).
Figure 24. The IR spectrum of DAPA (thin film on calcium fluoride plate) showed a strong IR band at 1784 cm-1 (5.6 mm), consistent with the expected absorbance for a carbonyl stretching in a fused b-lactam. The band at 1147 cm-1 (8.7 mm) is consistent with the presence of a sulfonamide bond linking the penicillanic acid amine group to the dansyl chromophore.
Mass spectrometry (FT-ICR MS) confirmed the compound to have MH+ of 450, in
accordance with DAPA calculated formula weight 450.11 (Fig. 25). 54
Figure 25. FT-ICR mass spectrum of DAPA showed a molecular ion of 450 amu, consistent with the proposed structure.
1H NMR chemical shifts 1.35(3H, s), 1.4(3H, s), 2.8(6H, s), 4.1(1H, d), 5.1(1H, d),
5.45(1H, s), 7.2(1H, d), 7.55(1H, d), 7.6(1H, dd), 8.2(1H, d), 8.3(1H, d), 8.4(1H, d) agreed with predicted values (Fig. 26).
4.2.4 Acylated Commercial Lactams
Acylation reactions were monitored by TLC eluted with 3:1:1 n-butanol:acetic acid:water.
Bioautography was used to monitor antibiotic activities of starting materials and products (Fig.
27). Typically approximately 1 to 5 microliter volumes of concentrated standards or reaction 55
extracts were applied to TLC plates; overloading caused streaks and was avoided in order to allow qualitative comparisons of antibiotic activities. The ninhydrin reaction and iodine development carried out on replicate thin-layer chromatograms permitted visual detection of reaction components. Acylated products were less polar than the underivatized amine starting materials.
Figure 26. 1H NMR spectrum of DAPA. Chemical shifts (35(3H, s), 1.4(3H, s), 2.8(6H, s), 4.1(1H, d), 5.1(1H, d), 5.45(1H, s), 7.2(1H, d), 7.55(1H, d), 7.6(1H, dd), 8.2(1H, d), 8.3(1H, d), 8.4(1H, d)) agreed with predicted values.
Initial bioassay evaluations showed ampicillin to have much stronger antibiotic activity than any compound extracted from its acylation reaction. Approximately one microliter of concentrated ampicillin standard solution run by TLC yielded a >2 cm diameter cleared zone on 56
the bacterial lawn corresponding to an Rf =0.4. There was also concern that acylations on this molecule were sterically hindered by the bulky aromatic group in proximity to the amine. Due to ineffectiveness of the reaction, as well as ambiguities the bioactivity of the starting material would introduce into quantitative determinations of reaction products later, ampicillin was discarded as a derivitization target.
Figure 27. Bioautography on lawns of B. subtilis was used to monitor acylation reactions on â-lactam starting materials. 57
Aminopenicillanic acid (APA) was well-resolved by TLC from its acetylated and
trifluoroacetylated derivatives (Rf = 0.25, 0.59, 0.71, respectively). It typically showed no activity
for quantities tested by bioautography. Acetylated APA, in contrast, cleared a zone of >1 cm in
diameter, and comparable amounts of trifluoroacetylated APA also cleared a zone of 1 to 2 cm.
APA acetylations succeeded under aqueous conditions outlined in the Materials and Methods
section.
Aminocephalosporanic acid (ACA) travelled on TLC with an Rf = 0.20, and showed little
(<5 mm diameter) to no zone of bioactivity. The acetylated product (Rf = 0.35) and the
trifluoroacetylated product (Rf = 0.56) yielded cleared zones of 1.5 to 2 cm in diameter in
quantities tested. ACA was more difficult to solubilize than APA. Starting material dissolved
more readily in chloroform than aqueous bicarbonate, thus more successful acetylation reactions
were performed with the organic system described in Materials and Methods.
1H NMR spectroscopy was used to ensure that the reaction products monitored by TLC were accurately identified. NMR examination did verify that the products isolated still contained the appropriate b-lactam core ( 1H NMR chemical shifts: APA 1.45(3H, s), 1.5(3H, s), 3.6(1H, s), 3.9(1H, b), 5.4(1H, d); ACA 2.3(3H, s), 3.7(1H, d), 3.8(1H, d), 4.9(1H, d), 5.1(1H, d),
5.15(1H, d), 5.2(1H, d)). The products were clearly modified as evidenced by TLC analysis.
Quantitative biological assays performed on M. fortuitum by serial dilution in 96-well microtiter plates were read on day 3 or 4 of incubation. Plates were frequently kept for up to a week after reading to see whether growth patterns changed; at times slow regrowth changed the reading by one or at most two dilutions. Day 4 readings were determined to yield the most clear and reproducible results. 58
Amikacin was used as a control antibiotic. MIC values for amikacin tested against mycobateria are reported to fall in the range from 1 to 12.5 micrograms per milliliter, with the average MIC of susceptible MTB being 1 microgram per milliliter.15 Amikacin MIC values of M. fortuitum determined in this lab ranged from 0.4 to 8.2 mg/mL.
Figure 28. Minimum inhibitory concentrations of acylated lactams measured for M. fortuitum.
Minimum inhibitory concentrations for antibiotically active compounds are typically reported in units of mass per volume (micrograms/mL). Because our interest was in comparing the effects of a specific modification on each compound tested, data is of more interest here when expressed as quantity of molecules per volume (micromoles/mL). Average values obtained for the inhibitory concentration of APA, acetylated APA (AcAPA), trifluoroacetylated APA
(TFAcAPA), ACA, acetylated ACA (AcACA), and trifluoroacetylated ACA (TFAcACA) did 59
indicate the possibility that substitution of fluorine for hydrogen in an equivalent b-lactam molecule may be capable of enhancing antimycobacterial activity (Fig. 28). This finding is consistent with other data from this lab which have suggested that trifluoroacylated monocyclic lactam products possess more apparent antimycobacterial activity in qualitative bioassays (L.
Anderson, D. Cross, R. Day, unpublished), as well as in quantitative MIC assays (D. Cross, R.
Day, unpublished).
4.3 PBPs and Protein Complexes
4.3.1 Labeling
Initial labeling experiments were carried out using crude material from the fluorinated, dansylated monocyclic lactam synthesis. At this time, all protein analysis was performed by RPLC with little success. This protocol proved problematic for several reasons. First, no subcellular fractionation was performed on cell lysate; thus proteins were at low concentrations.
Second, an effective protocol for elution of mycobacterial proteins under reversed-phase conditions was not developed. Trials using acetonitrile/water as mobile phase and trifluoroacetic acid as an ion-pairing reagent were unsuccessful. After multiple trials, the column was flushed with trifluoroethanol in order to dislodge any protein adsorbed to the hydrophobic stationary phase. During these cleaning runs, peaks which potentially contained protein of interest were detected. Peaks with strong absorbances at 320 nm were observed during the washing step, indicating that perhaps proteins which had the dansylated group covalently attached had accumulated on the column over several runs. Elution patterns were irregular, however, and could not be relied upon for analytical or preparative purposes. 60
This evidence led to attempts at using mobile phases with higher eluotropic strengths to
resolve sample proteins. Membrane proteins, which tend to be more hydrophobic than cytosolic
or secretory proteins, have been successfully eluted from reversed-phase columns using solvent
systems containing isopropanol, acetic acid, or formic acid. After acetonitrile/H2O elution
systems proved unsuccessful during attempts to separate membrane proteins of interest on C18 and C4 columns, mobile phases composed of isopropanol and formic acid were tested. No suitable elution conditions were designed.
The third problem with this series of experiments was the ambiguity as to whether mycobacterial proteins had actually been labeled in detectable quantities. Our lack of success was difficult to remedy without addressing all three of the compounded problems explained above.
To address this roadblock, several changes were made in the labeling protocol in order to better define the fluorescent labeling reagent, enrich the samples in our target proteins, and to adequately separate proteins. Ultracentrifugation of cell lysate at 100,000 x g allowed separation of the membrane pellet from cytosolic proteins, and provided a simple means of concentrating membrane protein into very small sample volumes. The membrane proteins were again assayed by RPLC, but once more proved intractable to this method. Polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) was found to afford a means of solubilization and separation of membrane proteins which could be adapted to our needs. And finally, DAPA was substituted for crude monocyclic lactam label, since it could be more readily characterized and obtained in pure form. DAPA was shown to label proteins in mycobacteria when used in conjunction with the b-lactamase inhibitor sulbactam (Fig. 29, 30). Successful labeling was demonstrated in DAPA/sulbactam treated M. aurum cells; electrophoretic separation of cell 61
proteins revealed that certain bands fluoresced strongly under ultraviolet light. DAPA appeared as a low molecular weight band when electrophoresed in a lane with molecular weight markers in a control experiment (Fig. 31). This control demonstrated that free, non-protein bound DAPA would not interfere with the detection of DAPA-labeled bacterial proteins of interest, which traveled in molecular weight ranges above approximately 60 kilodaltons.
Figure 29. M. aurum proteins isolated from DAPA-treated cells. After subcellular fractionation, membrane proteins were phase partitioned into aqueous- (peripheral membrane proteins, “aq”) and detergent- (integral membrane proteins, “TX-114”) soluble portions. PAGE gel stained with Coomassie blue to visualize proteins in all fractions—membrane proteins in lanes 2-7, cytosolic proteins in lanes 8-10.
1. MW standards 116, 97, 66, 29 kDa.; 2.aq. membrane fraction, no labeling treatment; 3. aq. membrane protein, DAPA ; 4. aq. membrane fraction, DAPA/sulbactam; 5. TX-114 fraction, no label 6. TX-114 fraction, DAPA; 7. TX-114 fraction, DAPA/sulbactam; 8. cytosol, no label; 9. cytosol, DAPA; 10. cytosol, DAPA/sulbactam.
62
Figure 30. PAGE gel of M. aurum proteins identical to that shown in figure 29 was electroblotted onto PVDF membrane. DAPA-labeled proteins fluoresce under UV light. Notice that strongest fluorescence is visible in lane 7, a sample of detergent-soluble membrane proteins from cells which have been DAPA/sulbactam treated. Coomassie staining of residual protein left in the polyacrylamide after blotting indicated that cytosolic proteins transferred more efficiently than membrane proteins (not shown).
1. MW standards 116, 97, 66, 29 kDa.; 2.aq. membrane fraction, no labeling treatment; 3. aq. membrane protein, DAPA ; 4. aq. membrane fraction, DAPA/sulbactam; 5. TX-114 fraction, no label 6. TX-114 fraction, DAPA; 7. TX-114 fraction, DAPA/sulbactam; 8. cytosol, no label; 9. cytosol, DAPA; 10. cytosol, DAPA/sulbactam.
63
Figure 31. DAPA appeared as a low molecular weight band (lower right-hand corner of PAGE gel) when electrophoresed in a lane with molecular weight markers in a control experiment. This control demonstrated that free, non-protein bound DAPA would not interfere with the detection of DAPA-labeled bacterial proteins of interest, which traveled in molecular weight ranges above approximately 60 kilodaltons.
4.3.2 Cell Fractionation, Protein Quantitation, and Phase Partition
The French pressure cell was an efficient means of mycobacterial cell lysis, but this
equipment was not available throughout the work. All membrane proteins subjected to
electrophoresis were obtained from cells lysed by sonication.
Mehotra estimated recovery of 3 mg membrane protein per gram of wet MTB cells.47 Our modified Lowry assay indicated that we achieved 25% of this recovery at best. Each liter of culture yielded 2-3 g of mycobacterial cells; approximately 1.5 mg of total membrane protein was recovered from this cell mass. After phase partitioning, membrane proteins were distributed in roughly comparable amounts between the detergent phase and the aqueous phase. M. aurum cells
yielded two to three times as much membrane protein per liter of culture.
Fluorescence appeared to partition into Triton X-114 during phase partitioning of
proteins. Control partitions performed with DAPA alone showed that while this fluorescent label 64
did dissolve to some extent in both phases, fluorescence appeared to be stronger in the detergent phase.
4.3.3 Electrophoresis and Electroblots
Samples solubilized under standard conditions of SDS and heat performed far better during PAGE and Western blotting than those treated with conditions customized for membrane proteins. Samples treated with higher concentrations of SDS and warmed to 40°C for 30 minutes or boiled briefly tended to streak upon blotting to polyvinylidene fluoride (PVDF) membranes, which resulted in poor resolution (Fig. 32).
Figure 32. PVDF electroblots of MTB membrane proteins from PAGE gels, seen under UV light. Samples had been prepared for electrophoresis in 3% SDS solubilization buffer, in contrast to 0.4% SDS standard buffer. Resolution was poor.
65
Fluorescence of the dansyl group was indistinct but visible in polyacrylamide slab gels inspected under UV light. In order to reliably detect all fluorescent bands, and to isolate proteins from the gel matrix, electroblots were typically performed. Three hours of blotting at 250-300 mAmps was sufficient for transfer of proteins to PVDF (Fig. 33).
Figure 33. Progress of electroblotting from PAGE gels was checked by removing PVDF strips from the blotting cassette at timed intervals. From right to left, strips were removed at 30, 60, 120, 180, and 220 minutes. Fluorescent proteins were detected under UV light.
Lane 1. MW standards, not visible under UV light. Lanes 2, 4, 6, 8, 10. Detergent-solubilized fraction of M. aurum membrane proteins labeled with DAPA/sulbactam, 96 mg protein per lane. Lanes 3, 5, 7, 9. Detergent- solubilized fraction of M. aurum membrane proteins labeled with DAPA/sulbactam, 48 mg protein per lane.
4.3.4 Protein Molecular Weight Estimation
The molecular weights of proteins exhibiting fluorescence after labeling treatments were estimated by comparing to bands of protein molecular weight standards. To obtain more accurate MW data, five distinct fluorescent bands having apparent MWs of 66 kDa (one band), 66
and 82-85 kDa (four bands) were selected on PVDF electroblot membranes and excised (Fig. 34).
The PVDF fragments were submitted for MALDI MS, but proved intractable to this analysis.
Figure 34. Lower photo: PVDF electroblot of PAGE-separated MTB proteins. DAPA was detected in six lanes under UV light; lanes showing fluorescence had membrane protein (outer four) and cytosolic protein (inner two) samples. Five distinct fluorescent bands having apparent MWs of 66 kDa (one band), and 82-85 kDa (four bands) are indicated.
Upper photo: Molecular weight standards were stained on PVDF by cutting off strips of the membrane and treating with Coomassie blue. 67
Molecular weight standards stained with Coomassie Blue or visualized by transillumination on PVDF were therefore taken as references to estimate the sizes of tagged proteins. Two-dimensional gel electrophoresis of crude membrane protein isolates confirmed that the target bands likely contained more than one protein species each. One dimensional gels, however, were run in order to separate fluorescent proteins into visible bands which could be extracted from polyacrylamide for analysis.
Figure 35. PVDF blot of MTB proteins, fluorescence visible under UV illumination. Cells were treated with sulbactam (4 mg/mL) and DAPA (8 mg/mL) for five hours.
Lanes 1 and 10: MW standards, no label. Lanes 2, 5, 8: membrane fractions from labeled control cells (no cyanogen). Lanes 3, 6, 9: membrane fraction from cells treated with cyanogen for five minutes. Lanes 4 and 7: cytosolic fractions from control and cyanogen-treated cells, respectively.
4.3.5 Cyanogen Cross-Linking
The treatment of cells with cyanogen, a quick-acting gaseous reagent, posed no technical problems. In labeled protein samples from cells not subjected to cyanogen, the strongest 68
fluorescence was observed in bands corresponding to molecular weight of approximately 66 kDa.
Each lane in which membrane protein from cyanogen-treated cells had been applied showed a single, strongly fluorescent band that had not migrated beyond the sample well edge of the stacking gel (Fig. 35, 36).
Figure 36. Enlargement from figure 35. PVDF blot of MTB proteins, fluorescence visible under UV illumination. Cells were DAPA/sulbactam labeled. Notice the single strongly fluorescent band which had not migrated beyond the well edge in lane 2, a cyanogen-treated sample.
1.MW standards, no label. 2. membrane fractions from labeled control cells (no cyanogen). 3. membrane fraction from cells treated with cyanogen for five minutes. 4. cytosolic fractions from control cells.
The cytosolic proteins from control and cyanogen-treated MTB cells were electrophoresed in separate lanes. One band in each of these lanes fluoresced under UV illumination. This protein apparently bound the DAPA label, but did not fractionate--at least not 69
entirely--with the plasma membrane. Interestingly, this cytosolic band had diminished
fluorescence in the cyanogen-treated sample. One explanation for this observation is that the
band corresponds to a PBP which is associated to the plasma membrane via hydrophobic
interaction, rather than a membrane-spanning domain. This type of interaction would be
disrupted during fractionation of cells. If the protein were cross-linked to other integral
membrane proteins before cell disruption, however, we would expect to see less of it in cytosolic
fractions.
4.3.6 Mass Spectrometry of Peptide Digests
The purpose of digesting samples with the site-specific enzymes trypsin and chymotrypsin
is to create a collection of peptides whose masses are in essence a fingerprint of the unidentified
parent protein. Molecular weight data from the digests of unknown proteins can be searched
against virtual digests of protein sequence databases available on the world wide web. Such
“peptide-mass fingerprinting” can provide a useful tool for making quick identifications of
unknown proteins.65 66 67 68
In order to recover fractions enriched in M. tuberculosis PBPs, polyacrylamide gels were
examined under UV light immediately after electrophoresis of protein extracted from dansyl-
APA-treated cells, and fluorescent bands were located. Bands which corresponded to proteins of
interest were excised from at least 6 lanes each, then gel slices from each distinct region were
65 Pappin, D., Hojrup, P., and Bleasby, A. (1993) Curr. Biol. 3, 327-332. 66 Henzel, W., Billeci, T., Stults, J., Wong, S., Grimley, C., Watanabe, C. (1993) Proc. Nat. Acad. Sci. USA 90, 5011-5015. 67 Mann, M., Højrup, P., Roepstorff, P. (1993) Biol. Mass Spec. 22, 338-345. 68 James, P., Quadroni, M., Carafoli, E., Gonnet, G. (1993) Biochem. Biophys. Res. Comm. 195, 58-64. 70
pooled separately and subjected to in-gel proteolytic digestion as described in Materials and
Methods.
Peptides by mass spectrometry requires gentle or “soft” ionization techniques which can
create charged gas phase ions without fragmentation. Two different instruments were used to
examine peptide digests isolated from sample polyacrylamide slices: one equipped with matrix-
assisted laser desorption ionization (MALDI) and the other using electrospray ionization (ESI).
Both instruments accomplish “soft” ionization of peptides without fragmentation, and both use
time-of-flight (TOF) mass analyzers to separate analyte ions.
MALDI-TOF data confirmed that bovine serum albumin was a component of all in-gel
digests (data not shown). This contamination presumably occurred due to the inclusion of bovine
serum albumin (an ingredient of Middlebrook ADC supplement) in all mycobacterial growth
media. Albumin was also found in a control slice of gel, which had been excised from the bottom
of a non-cyanogen-treated sample lane, and which showed no fluorescence. This demonstrated
that albumin contamination did not interfere with our identification of proteins of interest due to
non-specific DAPA binding.
ESI-Q-TOF data were entered into the MS-Fit 3.3.1 program (copyright 1995-2000, The
Regents of the University of California) available through the Protein Prospector website
managed by University of California at San Francisco (http://prospector.ucsf.edu).
The Sanger Centre, which stores the entire genomic database of MTB for public access, provides
a record of putative gene products classified by homology. Twelve sequences within the MTB
genome have been identified as putative PBP-encoding regions (Appendix 1.a). The Sanger
database, however, is not directly searchable using MS-Fit. The twelve entries of interest from
Sanger were therefore subjected to virtual tryptic digestion, and the results were used to search 71
the database of the National Center for Biotechnology Information. Initial queries were
performed on all mycobacterial genomic sequences stored in the NCBI database
(http://www3.ncbi.nlm.nih.gov) to learn which sequences here corresponded to the Sanger
records of interest. In most cases, searches matched to M. leprae proteins which have been
previously linked to M. tuberculosis homologs.
The NCBI accession numbers for these proteins were used in MS-Fit searches to find peptides in putative mycobacterial PBPs that match m/z signals found for actual sample digests.
Although this system does not unambiguously identify unknown proteins, it suits our purpose of tentatively assigning identities to PBPs which we have DAPA-labeled and separated by PAGE.
Protein of Estimated Molecular Weight 66 kDa
Trypsin and chymotrypsin digests of the fluorescent protein band of ~66kDa yielded ESI-
Q-TOF signals consistent with peptides from M. leprae PBP 2 (listed as “pbpB” by Sanger). This
protein is documented by the Sanger Centre to have 80.6% identity to the 72.4 kDa MTB protein
pbpB (Rv2163c) (Appendix 1.b).
Protein of Estimated Molecular Weight 82-85 kDa
Tryptic digestion of the labeled 82-85 kDa protein sample produced peptides which matched in mass to peptides from the M. leprae pon1, a class A penicillin binding protein. The
84.6 kDa MTB homolog ponA’ (Rv3682) is described in the Sanger Centre’s annotation as
“almost identical to…pon1=PBP1 Mycobacterium leprae [sic]” (Appendix 1.c). No chymotryptic
data were available.
72
High Molecular Weight Complex
Proteolytic digestion of proteins cross-linked by cyanogen produced peptides matching calculated peptide masses from three M. leprae proteins: one corresponding to MTB protein ponA (Rv0050), and two corresponding toMTB pbpA (Rv0016c). These MTB penicillin binding proteins have molecular weights of 71.1 and 51.6 kDa, respectively (Appendix 1.d).
4.3.7 Competitive Binding of Putative PBPs
Competitive binding assays were performed to confirm specificity of binding for DAPA, our fluorescent tag. MTB membrane fractions which were pre-treated with a combination of penicillin G (benzyloxypenicillin) and sulbactam, then labeled with DAPA, showed diminished fluorescence compared to similar amounts of membrane protein which were not pre-treated with unlabeled lactam. The effect can be seen on the protein band at a molecular weight of ~66 kDa
(Fig. 37). This result is a critical demonstration of the binding of fluorescent probe to the target
PBPs.
73
Figure 37. DAPA-labeled MTB membrane proteins in polyacrylamide under UV illumination. The major fluorescent band in non-cyanogen treated samples is diminished by pre-treatment with non-fluorescent competitor.
Lane 2: 88 mg protein from cyanogen-treated cells. Lane 3: 56 mg protein from cells pre-treated with penicillin G/sulbactam, then labeled with DAPA/sulbactam. Lanes 4 and 5: 52 mg and 66 mg of protein, respectively, from DAPA/sulbactam-labeled control cells (no competitor). Lane 6: duplicate of lane 2 with 20% smaller load of protein sample.
74
5. CONCLUSION
Our research objective centered on isolating membrane proteins from M. tuberculosis, an
important human pathogen. Specifically, we intended to develop a protocol for treating whole
MTB cells with fluorescent â-lactam, so that we could cross-link these proteins in native enzyme
complexes with cyanogen treatment of intact cells.
We found that by modifying the commercially available 6-aminopenicillanic acid with a fluorescent dansyl tag, we could make a probe capable of labeling PBPs in mycobacteria. This dansylated affinity label (DAPA) was chosen over the de novo monocyclic lactam which had been designed to penetrate mycobacteria and to be resistant to â-lactamase. The former fluorescent lactam proved to be far more easily characterized. The effectiveness of the DAPA label in mycobacteria depended on the addition of sulbactam, a â-lactamase inhibitor, to labeling treatments. Competition assays confirmed specificity of binding for DAPA to PBPs.
The major purpose of this work was to demonstrate that cyanogen can be used to cross- link PBPs in native protein complexes within intact mycobacterial cells. We accomplished this goal in whole cells from attenuated M. tuberculosis cultures. Cell lysate fractions successfully enriched for membrane proteins were separated by SDS-PAGE and electroblotted onto PVDF membranes in order to detect cross-linking of fluorescent PBPs.
Researchers have previously observed four major PBPs by using affinity labels on mycobacterial cell lysate fractions.1 2 Our method has not yet allowed full resolution and characterization of labeled PBPs, but our advantage has been the successful use of label in intact cells. We feel that this method will provide a guide for performing further cross-linking studies on mycobacterial PBPs, and for potentially isolating proteins of interest. The cross-linking studies 75
documented here offer a foundation for later investigations into the enzymology of mycobacterial cell wall biosynthesis.
76
Appendix 1.a. The Sanger Centre database of MTB proteins which have a role in cell envelope biosynthesis (http://www.sanger.ac.uk). Twelve penicillin binding sequences have been classified by homology.
77
II.C.3 Murein sacculus and peptidoglycan [28]
Gene Product pbpA Rv0016c penicillin-binding protein rodA Rv0017c FtsW/RodA/SpovE family ponA Rv0050 penicillin-bonding protein murB Rv0482 UDP-N-acetylenolpyruvoylglucosamine reductase ----- Rv0907 probable penicillin binding protein glmU Rv1018c UDP-N-acetylglucosamine pyrophosphorylase lytB' Rv1110 very similar to LytB murA Rv1315 UDP-N-acetylglucosamine-1-carboxyvinyltransferase murI Rv1338 glutamate racemase ----- Rv1367c probable penicillin binding protein ----- Rv1730c probable penicillin binding protein ----- Rv1922 probable penicillin binding protein murC Rv2152c UDP-N-acetyl-muramate-alanine ligase murG Rv2153c transferase in peptidoglycan synthesis murD Rv2155c UDP-N-acetylmuramoylalanine-D-glutamate ligase murX Rv2156c phospho-N-acetylmuramoyl-pentapeptide transferase murF Rv2157c D-alanine:D-alanine-adding enzyme murE Rv2158c meso-diaminopimelate-adding enzyme pbpB Rv2163c penicillin-binding protein 2 ----- Rv2864c probable penicillin binding protein dacB Rv2911 penicillin binding protein ddlA Rv2981c D-alanine-D-alanine ligase A ----- Rv3330 probable penicillin binding protein nagA Rv3332 N-acetylglucosamine-6-P-deacetylase lytB Rv3382c LytB protein homologue ----- Rv3627c probable penicillin binding protein ponA' Rv3682 class A penicillin binding protein glf Rv3809c UDP-galactopyranose mutase
78
Appendix 1.b. Peptide digest data from MTB PBP of estimated molecular weight ~66 kDa by SDS-PAGE. ESI- Q-TOF data was submitted as m/z; peptide ion masses were calculated by MS-FIT / Protein Prospector software found at http://prospector.ucsf.edu. Programs were developed in the UCSF Mass Spectrometry Facility, which is directed by Dr. Alma Burlingame, Professor of Chemistry and Pharmaceutical Chemistry at UCSF.
Protein sequences and annotation courtesy of the Sanger Centre (http://www.sanger.ac.uk).
79
Result Summary: Tryptic Digest of ~66kDa Band
# (%) Protein Accession Masses Species Protein Name MW (Da)/pI # Matched (AL583925) MYCOBACTERIUM 13/91 (14%) 84511.8 / 7.00 13093933 penicillin binding LEPRAE protein (class A) (AL022602) MYCOBACTERIUM 10/91 (10%) 72422.2 / 9.23 3080479 penicillin binding LEPRAE protein 2
Detailed Results
13/91 matches (14%). 84511.8 Da, pI = 7.00. Acc. # 13093933. MYCOBACTERIUM LEPRAE. (AL583925) penicillin binding protein (class A) .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 416.9413 416.2985 0.6428 167 169 (R) KLR(E) 416.9413 417.2462 -0.3049 170 172 (R) EIR(I) 494.0517 493.2775 0.7742 425 428 (R) FQAK(G) 545.1309 544.3571 0.7738 716 719 (R) QRLK(G) 664.5376 664.3088 0.2288 429 436 (K) GMGSGGAK(G) 699.9939 700.3994 -0.4055 419 424 (K) LEVPSR(F) 795.4478 795.4365 0.0113 687 693 (R) LPPTDPR(Y) 820.5164 820.3623 0.1541 287 294 (R) GCIAAGDR(A)
80
10/91 matches (10%). 72422.2 Da, pI = 9.23. Acc. # 3080479. MYCOBACTERIUM LEPRAE. (AL022602) penicillin binding protein 2 .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 416.9413 416.2985 0.6428 157 159 (K) KIR(Q) 483.0509 482.3091 0.7418 520 523 (R) IPPR(I) 545.1309 544.3459 0.7850 561 565 (R) AVVQK(D) 547.0455 547.3026 -0.2571 557 560 (R) QMLR(A) 582.6047 582.3225 0.2822 296 299 (R) NRHR(A) 647.9473 648.3317 -0.3844 132 137 (R) GSITDR(N) 769.9494 770.4525 -0.5031 32 37 (R) LRQPEK(A) 830.9929 830.3871 0.6058 454 459 (R) FYDMVR(K)
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Result Summary: Chymotryptic Digest of ~66kDa Band
Protein Accession Species Protein Name MW (Da)/pI # (AL022602) MYCOBACTERIUM 72422.2 / 9.23 3080479 penicillin binding LEPRAE protein 2 (AF165523) MYCOBACTERIUM 74459.4 / 4.59 10121238 penicillin-binding SMEGMATIS protein 1 MYCOBACTERIUM (AF017098) 21690.5 / 6.03 2736089 TUBERCULOSIS pGB14T-P
Detailed Results
72422.2 Da, pI = 9.23. Acc. # 3080479. MYCOBACTERIUM LEPRAE. (AL022602) penicillin binding protein 2 .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 203.0838 203.1396 -0.0558 93 94 (F) AL(M) 393.0131 393.2138 -0.2007 103 105 (L) FNL(Q) 394.0044 394.2342 -0.2298 210 212 (F) VYL(A) 566.7556 566.2979 0.4577 608 611 (Y) WITF(A) 625.5483 625.3197 0.2286 429 434 (Y) TTTGVF(G) 789.0370 789.4470 -0.4100 145 151 (F) TIESRAL(T) 795.4478 795.4187 0.0291 456 461 (Y) DMVRKF(G) 796.4532 796.4106 0.0426 648 654 (F) HNVAGWL(M) 830.9929 830.4048 0.5881 479 485 (L) VPSVDQW(S) 830.9929 830.3949 0.5980 647 653 (L) FHNVAGW(L)
82
74459.4 Da, pI = 4.59. Acc. # 10121238. MYCOBACTERIUM SMEGMATIS. (AF165523) penicillin-binding protein 1 .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 203.0838 203.1396 -0.0558 362 363 (F) AL(V) 393.0131 393.2502 -0.2371 359 361 (F) KVF(A) 394.0044 394.1978 -0.1934 276 278 (L) DLF(D) 566.7556 567.2779 -0.5223 481 486 (L) AASGVY(H) 794.9279 794.3473 0.5806 87 93 (Y) SNPGFSW(T)
21690.5 Da, pI = 6.03. Acc. # 2736089. MYCOBACTERIUM TUBERCULOSIS. (AF017098) pGB14T-P .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 203.0838 203.1396 -0.0558 142 143 (F) AL(R) 789.0370 789.3267 -0.2897 196 202 (F) DDTLNDP(-) 795.4478 795.4041 0.0437 167 172 (Y) LYHYSL(D)
83
pbpB
Gene name: Class: Start/End: Length: Molecular weight: pbpB ML0908 II.C.3 1073832 / 1075859 2028 bp / 675 aa 72422 Da Product: penicillin-binding protein 2
Description:
Identical to the previously sequenced Mycobacterium leprae penicillin binding protein 2 TR:O69558 (EMBL:AL022602) (675 aa); Fasta score E(): 0, 99.9% identity in 675 aa overlap(EMBL:AL022602). Also highly similar to Mycobacterium tuberculosis hypothetical protein TR:O06214 (EMBL:Z95388) (679 aa); Fasta score E(): 0, 80.6% identity in 677 aa overlap(EMBL:Z95388) and to many other penicillin-binding proteins e.g. Neisseria meningitidis SW:PBP2_NEIME (P11882) (581 aa); Fasta score E(): 4.3e-29, 32.4% identity in 590 aa overlap(P11882). Contains Pfam match to entry PF00905 Transpeptidase, Penicillin binding protein transpeptidase domain. Contains PS00017 ATP/GTP-binding site motif A (P-loop).
Translation: MRRGDAHRPC SSRSAQLGNS SSTPPVRQPK RLRQPEKARK AKDTKKSRSA VAADAVTEGR SARKRRTRQV VEIGTYGPSF IFRHRGGNVV IFALMLVAAT QLFNLQVSNA AGLRAQAAGQ LRVTDVEKAV RGSITDRNNE QLAFTIESRA LTFQPKKIRQ QLEEAKQKTP SAPDPQQRLQ DIAKEVAGRL SNKPDGPSLL KKLQSNDSFV YLARAVDPAV AEAISAKYPE VGSERQDLRQ YPGGSLAANI VGGIDWDGHG LLGLEDSLDS VLSGTDGSVT YDRGSDGVVI PGSYRNRHRA VNGSTVQLTI DDDIQFYVQQ QVQQAKNLSG AHNVSAVVLD AKTGEVLAMA NDNTFDPSQD IGRQGGKQLG NLAVSSPFEP GSVNKVITAS AVIEYGLSTP DEVLQVPGSI QMGGVSVHDA WEHGVMPYTT TGVFGKSSNV GTLMLAQRVG PERFYDMVRK FGLGQLTGVG LPGESEGLVP SVDQWSGSTF SNLPIGQGLS TTLLQMTGMY QVIANDGVRI PPRIIKATNA ADGTRTQEPR PDGIRVVSPQ TAQTVRQMLR AVVQKDPMGY QQGTGPAAGV SGYQIAGKTG TAQQVNPACR CYFDDVYWIT FAGMATVDNP RYVIGTMMDN PERNADGTPG HSAAPLFHNV AGWLMQRENV PLSPDPGPPL TLQAT Pfam domains present: Pfam match to entry PF00905 Transpeptidase, Penicillin binding protein transpeptidase domain, score 417.90, E-value 9.6e-122 Prosite patterns present: PS00017 ATP/GTP-binding site motif A (P-loop)
84
pbpB
Gene name: Class: Start/End: Length: Molecular weight: pbpB Rv2163c II.C.3 2425051 / 2427087 2037 bp / 679 aa 72538 Da Product: penicillin-binding protein 2
Description:
Rv2163c, (MTCY270.05), len: 679. Function pbpB: probable penicillin-binding protein, similar to many bacterialPBP2s and to MTCY10H4.16c; 2.8e-10. Contains possible ATP/GTP-binding site motif A (P- loop; PS00017) near C-terminus.FASTA best: PBP2_NEIME P11882 penicillin-binding protein 2(pbp-2). (581 aa) opt: 665 z-score: 691.4 E(): 1.6e-31; (33.2% identity in 591 aa overlap)
Translation: MSRAAPRRAS QSQSTRPARG LRRPPGAQEV GQRKRPGKTQ KARQAQEATK SRPATRSDVA PAGRSTRARR TRQVVDVGTR GASFVFRHRT GNAVILVLML VAATQLFFLQ VSHAAGLRAQ AAGQLKVTDV QPAARGSIVD RNNDRLAFTI EARALTFQPK RIRRQLEEAR KKTSAAPDPQ QRLRDIAQEV AGKLNNKPDA AAVLKKLQSD ETFVYLARAV DPAVASAICA KYPEVGAERQ DLRQYPGGSL AANVVGGIDW DGHGLLGLED SLDAVLAGTD GSVTYDRGSD GVVIPGSYRN RHKAVHGSTV VLTLDNDIQF YVQQQVQQAK NLSGAHNVSA VVLDAKTGEV LAMANDNTFD PSQDIGRQGD KQLGNPAVSS PFEPGSVNKI VAASAVIEHG LSSPDEVLQV PGSIQMGGVT VHDAWEHGVM PYTTTGVFGK SSNVGTLMLS QRVGPERYYD MLRKFGLGQR TGVGLPGESA GLVPPIDQWS GSTFANLPIG QGLSMTLLQM TGMYQAIAND GVRVPPRIIK ATVAPDGSRT EEPRPDDIRV VSAQTAQTVR QMLRAVVQRD PMGYQQGTGP TAGVPGYQMA GKTGTAQQIN PGCGCYFDDV YWITFAGIAT ADNPRYVIGI MLDNPARNSD GAPGHSAAPL FHNIAGWLMQ RENVPLSPDP GPPLVLQAT Pfam domains present: Not available Prosite patterns present: PS00017 ATP/GTP-binding site motif A (P-loop)
85
Appendix 1.c. Peptide digest data from MTB PBP of estimated molecular weight of ~85 kDa by SDS-PAGE. ESI-Q-TOF data was submitted as m/z; peptide ion masses were calculated by MS-FIT / Protein Prospector software. Protein sequence and annotation were found in the Sanger Centre database (http://www.sanger.ac.uk).
86
Result Summary: Tryptic Digest of ~85kDa Band
rotein Accession Species Protein Name MW (Da)/pI # MYCOBACTERIUM (AL583925) penicillin binding 84511.8 / 7.00 13093933 LEPRAE protein (class A)
Detailed Results
84511.8 Da, pI = 7.00. Acc. # 13093933. MYCOBACTERIUM LEPRAE. (AL583925) penicillin binding protein (class A) . + Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 289.9669 290.1464 -0.1795 313 314 (K) DR(L) 664.0519 664.3088 -0.2569 429 436 (K) GMGSGGAK(G) 678.1337 678.3939 -0.2602 318 323 (K) GGYLIR(T) 820.5047 820.3623 0.1424 287 294 (R) GCIAAGDR(A)
87
pon1
Gene name: Class: Start/End: Length: Molecular weight: pon1 ML2308 II.C.3 2732187 / 2734598 2412 bp / 803 aa 84512 Da Product: penicillin binding protein (class A)
Description:
Previously characterised Mycobacterium leprae penicillin-binding protein Pon1 TR:P72351 (EMBL:S82044) (821 aa) fasta scores: E(): 0, 98.6% id in 783 aa. Contains a possible N-terminal signal sequence. Contains Pfam match to entry PF00905 Transpeptidase, Penicillin binding protein transpeptidase domain. Contains Pfam match to entry PF00912 Transglycosyl, Transglycosylase.
Translation: MSERLPAVLA VLKLAGYCLL AGVVTTAMMF PLAGGLGVIS NRASEVVANG SAQLLEGEVP AVSTMVDAKG NTIAWLYSQR RFEVPTDKIA NTMKLAIVSI EDKRFTDHNG VDWPGTLTGL AGYASGDVDT RGGSTIEQQY VKNYQLLVTA QTDAEKRAAV ETTPARKLRE IRIALTLDKT FTKPEILTRY LNLVSFGNNS FGVQDAAQTY FGVNASDLNW QQAALLAGMV QSTSTLNPYT NPEGALARRN LVLDTMIENL PQDAEALRAA KTEPLGILPR PNELPRGCIA AGDRAFFCDY VQEYLSHAGI SKDRLAKGGY LIRTTLDPNV QAPVKAAIDK FASPNLAGIS SVMSVITPGK DAHRVIAMGS NRRYGLDTEA GETMRPQTFS LVGDGAGSVF KIFTTAAALD MGMGINAKLE VPSRFQAKGM GSGGAKGCPK ETWCVINAGN YRGSMNVTDA LATSPNTAFA KLIQQVGVAR TVDMAIKLGL RSYTTPGTAR DYNPDSNESL ADFVKRQNIG SFTLGPIKVN ALELSNVAAT LASGGMWCPP NPIDKLFDRN GNEVAVTVET CDRVVPEGLA NTLANAMSKD TKGSGTAAGS AGAAGWDLPM SGKTGTTEAH RSSGFVGFTN HYAAANYIYD DSTSPTNLCS GPLRHCSNGD LYGGNEPART WFTAMKPIAT KFGEVRLPPT DPRYVDGSPG SQVPSVTGLD LDAARQRLKG AGFQVADQPT LINSTAKLGA VVGTTPSGQT IPGSIITIQT SSGIPPAPPP PPEGGPPMPP SVGSQVIEIP GLPPITIPLL APP Pfam domains present: Pfam match to entry PF00912 Transglycosyl, Transglycosylase, score 162.80, E- value 5.7e-45 Pfam match to entry PF00905 Transpeptidase, Penicillin binding protein transpeptidase domain, score -29.60, E-value 2.6e-05 Prosite patterns present: None
88
ponA'
Gene name: Class: Start/End: Length: Molecular weight: ponA' Rv3682 II.C.3 4121915 / 4124344 2430 bp / 810 aa 84636 Da Product: class A penicillin binding protein
Description:
Rv3682, (MTV025.030), ponA, len: 810. CLASS A PENICILLIN-BINDING PROTEIN, almost identical to gp|S82044|S82044_1 pon1=PBP1 Mycobacterium leprae,(821 aa), FASTA scores:opt: 4547 z-score: 3629.2 E(): 0, 88.0% identity in 769 aaoverlap. Also similar to MTCY21D4_13,28.1% identity in 662aaoverlap; MSGDNAB_6 Mycobacterium leprae cosmid L222, 26.6%identity in 661 aa overlap.
Translation: MPERLPAAIT VLKLAGCCLL ASVVATALTF PFAGGLGLMS NRASEVVANG SAQLLEGQVP AVSTMVDAKG NTIAWLYSQR RFEVPSDKIA NTMKLAIVSI EDKRFADHSG VDWKGTLTGL AGYASGDLDT RGGSTLEQQY VKNYQLLVTA QTDAEKRAAV ETTPARKLRE IRMALTLDKT FTKSEILTRY LNLVSFGNNS FGVQDAAQTY FGINASDLNW QQAALLAGMV QSTSTLNPYT NPDGALARRN VVLDTMIENL PGEAEALRAA KAEPLGVLPQ PNELPRGCIA AGDRAFFCDY VQEYLSRAGI SKEQVATGGY LIRTTLDPEV QAPVKAAIDK YASPNLAGIS SVMSVIKPGK DAHKVLAMAS NRKYGLDLEA GETMRPQPFS LVGDGAGSIF KIFTTAAALD MGMGINAQLD VPPRFQAKGL GSGGAKGCPK ETWCVVNAGN YRGSMNVTDA LATSPNTAFA KLISQVGVGR AVDMAIKLGL RSYANPGTAR DYNPDSNESL ADFVKRQNLG SFTLGPIELN ALELSNVAAT LASGGVWCPP NPIDQLIDRN GNEVAVTTET CDQVVPAGLA NTLANAMSKD AVGSGTAAGS AGAAGWDLPM SGKTGTTEAH RSAGFVGFTN RYAAANYIYD DSSSPTDLCS GPLRHCGSGD LYGGNEPSRT WFAAMKPIAN NFGEVQLPPT DPRYVDGAPG SRVPSVAGLD VDAARQRLKD AGFQVADQTN SVNSSAKYGE VVGTSPSGQT IPGSIVTIQI SNGIPPAPPP PPLPEDGGPP PPVGSQVVEI PGLPPITIPL LAPPPPAPPP Pfam domains present: Not available Prosite patterns present: None
89
Appendix 1.d. Peptide digest data from MTB PBPs which had been complexed by cyanogen. ESI-Q-TOF data was submitted as m/z; peptide ion masses were calculated by MS-FIT / Protein Prospector software. Protein sequences and annotation for M. leprae were located in the NCBI database and cross-referenced to MTB PBP records found in the Sanger Centre database(http://www.sanger.ac.uk).
90
Result Summary: Tryptic Digest of Cyanogen Complex
# (%) Protein Accession Rank Masses Species Protein Name MW (Da)/pI # Matched (AL583922) MYCOBACTERIUM 1 9/46 (19%) 131028.5 / 4.97 13093410 possible cell LEPRAE division protein (AL583917) MYCOBACTERIUM putative 2 4/46 (8%) 51756.1 / 6.18 13092428 LEPRAE penicillin- binding protein probable MYCOBACTERIUM 3 4/46 (8%) 50416.4 / 6.71 7476086 penicillin- LEPRAE binding protein (AL022118) MYCOBACTERIUM putative 4 4/46 (8%) 88572.4 / 9.04 2959415 LEPRAE penicillin- binding protein (AL022602) MYCOBACTERIUM 5 5/46 (10%) 72422.2 / 9.23 3080479 penicillin LEPRAE binding protein 2
Detailed Results
1. 9/46 matches (19%). 131028.5 Da, pI = 4.97. Acc. # 13093410. MYCOBACTERIUM LEPRAE. (AL583922) possible cell division protein . + Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 305.0791 304.1621 0.9170 365 366 (R) ER(H) 513.0325 513.3149 -0.2824 73 77 (R) APLGR(A) 588.0059 588.3105 -0.3046 821 826 (R) AAAAER(E) 615.9954 616.3782 -0.3828 175 179 (K) EKALR(K) 630.0028 630.3687 -0.3659 224 228 (R) DARLR(L) 646.9154 646.3888 0.5266 300 305 (R) VSATIR(I) 1035.8306 1035.6176 0.2130 170 176 (K) YRRRKEK(A) 1172.7184 1172.6751 0.0433 300 310 (R) VSATIRIAGER(A)
91
1172.7184 1172.6388 0.0796 475 484 (R) DAERQVVSLR(A) 1221.6134 1221.5963 0.0171 1192 1203 (R) GQQVESLVTSSS(-)
2. 4/46 matches (8%). 51756.1 Da, pI = 6.18. Acc. # 13092428. MYCOBACTERIUM LEPRAE. (AL583917) putative penicillin-binding protein .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 722.1237 721.3667 0.7570 1 6 (-)MNTSLR(R) 999.8017 999.5475 0.2542 482 492 (R) AVIEAALQGGA(-) 1082.3124 1082.5747 -0.2623 119 127 (R) RLADFFTGR(D) 1141.3544 1141.5238 -0.1694 426 436 (K) TGTAEHGTDPR(H)
3. 4/46 matches (8%). 50416.4 Da, pI = 6.71. Acc. # 7476086. MYCOBACTERIUM LEPRAE. probable penicillin-binding protein.
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 472.1295 472.2884 -0.1589 283 286 (R) VTPR(S) 722.1237 721.3667 0.7570 1 6 (-)MNTSLR(R) 1082.3124 1082.5747 -0.2623 119 127 (R) RLADFFTGR(D) 1141.3544 1141.5238 -0.1694 426 436 (K) TGTAEHGTDPR(H)
4. 4/46 matches (8%). 88572.4 Da, pI = 9.04. Acc. # 2959415. MYCOBACTERIUM LEPRAE. (AL022118) putative penicillin-binding protein .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 472.1295 471.2931 0.8364 78 81 (R) NPIK(E) 999.8017 999.5839 0.2178 78 86 (R) NPIKEVTAK(L) 1035.8306 1036.5097 -0.6791 67 76 (R) SALPTSMSSR(R) 1086.8047 1087.6085 -0.8038 91 101 (R) SAATVGGGRRR(R)
92
5. 5/46 matches (10%). 72422.2 Da, pI = 9.23. Acc. # 3080479. MYCOBACTERIUM LEPRAE. (AL022602) penicillin binding protein 2 .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 529.1721 528.3258 0.8463 28 31 (R) QPKR(L) 588.0059 588.3694 -0.3635 65 68 (K) RRTR(Q) 973.8471 974.5383 -0.6912 129 137 (K) AVRGSITDR(N) 1102.3221 1101.5904 0.7317 160 168 (R) QQLEEAKQK(T) 1201.0127 1201.7421 -0.7294 150 159 (R) ALTFQPKKIR(Q)
93
Result Summary: Chymotryptic Digest of Cyanogen Complex
# (%) Protein Accession Rank Masses Species Protein Name MW (Da)/pI # Matched (AL583926) MYCOBACTERIUM 1 6/34 (17%) 75662.7 / 7.13 13093882 penicillin-bonding LEPRAE protein (AL022118) MYCOBACTERIUM putative 2 6/34 (17%) 88572.4 / 9.04 2959415 LEPRAE penicillin-binding protein (L39923) MYCOBACTERIUM 3 5/34 (14%) 72803.3 / 5.98 886307 penicillin binding LEPRAE protein probable MYCOBACTERIUM 4 6/34 (17%) 50416.4 / 6.71 7476086 penicillin-binding LEPRAE protein (AL583917) MYCOBACTERIUM putative 5 6/34 (17%) 51756.1 / 6.18 13092428 LEPRAE penicillin-binding protein
Detailed Results
1. 6/34 matches (17%). 75662.7 Da, pI = 7.13. Acc. # 13093882. MYCOBACTERIUM LEPRAE. (AL583926) penicillin-bonding protein . + Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 295.1728 295.1658 0.0070 28 29 (L) YL(A) 382.2065 382.2454 -0.0389 12 14 (L) LHL(N) 382.2065 382.1978 0.0087 26 28 (Y) SLY(L) 397.1656 397.1723 -0.0067 182 184 (L) QSY(L) 437.0605 437.2512 -0.1907 23 25 (W) VRY(S) 557.2098 557.3299 -0.1201 655 660 (Y) AGVPTL(A)
94
2. 6/34 matches (17%). 88572.4 Da, pI = 9.04. Acc. # 2959415. MYCOBACTERIUM LEPRAE. (AL022118) putative penicillin-binding protein .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 295.1728 295.1658 0.0070 150 151 (L) YL(A) 382.2065 382.2454 -0.0389 134 136 (L) LHL(N) 382.2065 382.1978 0.0087 148 150 (Y) SLY(L) 397.1656 397.1723 -0.0067 304 306 (L) QSY(L) 437.0605 437.2512 -0.1907 145 147 (W) VRY(S) 557.2098 557.3299 -0.1201 777 782 (Y) AGVPTL(A)
3. 5/34 matches (14%). 72803.3 Da, pI = 5.98. Acc. # 886307. MYCOBACTERIUM LEPRAE. (L39923) penicillin binding protein .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 295.1728 295.1658 0.0070 6 7 (L) YL(A) 382.2065 382.1978 0.0087 4 6 (Y) SLY(L) 397.1656 397.1723 -0.0067 160 162 (L) QSY(L) 557.2098 557.3299 -0.1201 633 638 (Y) AGVPTL(A)
4. 6/34 matches (17%). 50416.4 Da, pI = 6.71. Acc. # 7476086. MYCOBACTERIUM LEPRAE. probable penicillin-binding protein.
m/z MH+ Delta Peptide Sequence start end submitted matched Da (Click for Fragment Ions)
95
382.2065 382.1978 0.0087 91 93 (F) YSL(R) 457.1369 457.3390 -0.2021 16 19 (L) IVLL(L) 458.1359 458.2251 -0.0892 183 186 (Y) DPNL(L) 499.0173 499.2193 -0.2020 121 124 (L) ADFF(T) 513.0140 513.2713 -0.2573 444 447 (W) YIAF(A) 529.1815 529.2986 -0.1171 373 377 (L) KGPDL(A)
5. 6/34 matches (17%). 51756.1 Da, pI = 6.18. Acc. # 13092428. MYCOBACTERIUM LEPRAE. (AL583917) putative penicillin-binding protein .
+ Peptide Sequence m/z MH Delta start end (Click for Fragment submitted matched Da Ions) 382.2065 382.1978 0.0087 91 93 (F) YSL(R) 457.1369 457.3390 -0.2021 16 19 (L) IVLL(L) 458.1359 458.2251 -0.0892 183 186 (Y) DPNL(L) 499.0173 499.2193 -0.2020 121 124 (L) ADFF(T) 513.0140 513.2713 -0.2573 444 447 (W) YIAF(A) 529.1815 529.3350 -0.1535 288 292 (F) VQLGL(L) 529.1815 529.2986 -0.1171 373 377 (L) KGPDL(A)
96
LOCUS CAA17957 830 aa BCT 27-AUG-1999 DEFINITION putative penicillin-binding protein [Mycobacterium leprae]. ACCESSION CAA17957 PID g2959415 VERSION CAA17957.1 GI:2959415 DBSOURCE embl locus MLCB1913, accession AL022118.1 KEYWORDS . SOURCE Mycobacterium leprae. ORGANISM Mycobacterium leprae Bacteria; Firmicutes; Actinobacteria; Actinobacteridae; Actinomycetales; Corynebacterineae; Mycobacteriaceae; Mycobacterium. REFERENCE 1 (residues 1 to 830) AUTHORS Eiglmeier,K., Honore,N., Woods,S.A., Caudron,B. and Cole,S.T. TITLE Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae JOURNAL Mol. Microbiol. 7 (2), 197-206 (1993) MEDLINE 93188700 REFERENCE 2 (residues 1 to 830) AUTHORS Murphy,L. and Harris,D. JOURNAL Unpublished REFERENCE 3 (residues 1 to 830) AUTHORS Parkhill,J., Barrell,B.G. and Rajandream,M.A. TITLE Direct Submission JOURNAL Submitted (06-MAR-1998) Mycobacterium leprae sequencing project, Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA E-mail: [email protected] Cosmids supplied by Dr. Stewart T. Cole, [3] Unite de Genetique Moleculaire Bacterienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France Requests for cosmids should be sent to Karin Eiglmeier ([email protected]) COMMENT Notes: The Sanger Centre is funded to complete the sequence of M. leprae by the Heiser Program for Research in Leprosy and Tuberculosis of The New York Community Trust. Work in Paris is supported by the Heiser Trust, the Association Francaise Raoul Follereau and the Groupement de Recherches et des Etudes des Genomes (GIP-GREG). Details of M. leprae sequencing at the Sanger Centre are available on the World Wide Web. (URL, http://www.sanger.ac.uk/Projects/) CDS are numbered using the following system eg MLCB33.01c. ML (M. leprae), cB33 (cosmid name), .01 (first CDS), c (complementary strand). The more significant matches with motifs in the PROSITE database are also included but some of these may be fortuitous. The length in codons is given for each CDS. Usually the highest scoring match found by fasta -o is given for CDS which show significant similarity to other CDS in the database. The position of possible ribosome binding site sequences are given where these have been used to deduce the initiation codon. All CDS 97
over 100 codons have been analysed. Gene prediction is based on positional base preference in codons especially where there is an increase in the observed/expected third position G + C. CAUTION: We may not have predicted the correct initiation codon. Where possible we choose an initiation codon (atg, gtg, or ttg) which is preceded by an upstream ribosome binding site sequence (optimally 5-13bp before the initiation codon). If this cannot be identified we choose the most upstream initiation codon. IMPORTANT: This sequence MAY NOT be the entire insert of the sequenced clone. It may be shorter because we only sequence overlapping sections once, or longer, because we arrange for a small overlap between neighbouring submissions. Cosmid B1913 overlaps EM_BA:MLDNAB L39923 Mycobacterium leprae cosmid L222 at the 3' end. FEATURES Location/Qualifiers source 1..830 /organism="Mycobacterium leprae" /db_xref="taxon:1769" /clone="cosmid B1913" Protein 1..830 /product="putative penicillin-binding protein" CDS 1..830 /gene="MLCB1913.23c" /db_xref="SPTREMBL:O53129" /coded_by="complement(AL022118.1:34393..36885)" /transl_table=11 /note="MLCB1913.23c, probable penecillin-binding protein, len: 830 aa; similar to many eg. TR:O05194 (EMBL:U80933) penicillin-binding protein 1, ponA from Neisseria meningitidis (798 aa), fasta scores; opt: 427, E(): 1.5e-25, 26.9% identity in 713 aa overlap" ORIGIN 1 msgpatervg erpssdqpgq rrqvplddrm taiipsatde rsagrvdple avkaaldgpp 61 pampfrsalp tsmssrrnpi kevtakldgr saatvgggrr rrlpsdppsp kqpgratgpg 121 wrvpgsrlnr twllhlnrqv nwrwvrysly lavvvllllp ivtftmtyfm vkvprpgdir 181 tnqvstilan ngleiakivp pegnrvdvnl sqvpvhvrqa viaaedrnfy snpgfdfras 241 vravqnnlfg sgdlqggsti tqqyvknalv gsaqhgfdgl mrktkelvia ikmsdawskd 301 dvlqsylnii yfgrgaygis aaakayfdkp veqltcsega llaalirrps vldpainlkg 361 vtarwnwvld gmvdinalsp ndrsvqlfpa tvppdqaraq nqttgpngli erqvvkelfe 421 lfnidrqtln tqglqvitti dpqaqrivek avskyldgqd pdmraaivsi dphngairay 481 yggadangfd faqaglqtgs sfkvfaliaa leqgiglgyr vdsspltvdg ikitnngdes 541 cgncniaeal klslntsyyr lmlklkngpq avadaahqag ivssfpgvah tlsedgkggp 601 pnngivlgqy etrvidmasa yatlaasgvy hrphlvqkvv naegrvlfda stadnsgdqr 661 iakavadnvt aamqpiagys rghnlaggrp saaktgtvql gdttankdaw mvgytpslst 721 avwvgtvkdn vplitasgea iygsglpsdi wkstmdgale gtpnegfpkp tevggyagvp 781 tlapppplkk viqptievap giiipvgppt tvtvpptqal pdlttsttpp
98
LOCUS CAC29526 492 aa BCT 20-FEB-2001 DEFINITION putative penicillin-binding protein [Mycobacterium leprae]. ACCESSION CAC29526 PID g13092428 VERSION CAC29526.1 GI:13092428 DBSOURCE embl locus MLEPRTN1, accession AL583917.1 KEYWORDS . SOURCE Mycobacterium leprae. ORGANISM Mycobacterium leprae Bacteria; Firmicutes; Actinobacteria; Actinobacteridae; Actinomycetales; Corynebacterineae; Mycobacteriaceae; Mycobacterium. REFERENCE 1 (residues 1 to 492) AUTHORS Cole,S.T., Eiglmeier,K., Parkhill,J., James,K.D., Thomson,N.R., Wheeler,P.R., Honore,N., Ganier,T., Churcher,C., Harris,D., Mungall,K., Basham,D., Brown,D., Chillingworth,T., Connor,R., Davies,R.M., Devlin,K., Duthoy,S., Feltwell,T., Fraser,A., Hamlin,N., Holroyd,S., Hornsby,T., Jagels,K., Lacroix,C., Maclean,J., Moule,S., Murphy,L., Oliver, Quail,M.A., Rajandream,M.-A., Rutherford,K.M., Rutter,S., Seeger,K., Simon,S., Simmonds,M., Skelton,J., Squares,R., Squares,S., Stevens,K., Taylor,K., Whitehead,S., Woodward,J.R. and Barrell,B.G. TITLE Massive gene decay in the leprosy bacillus JOURNAL Nature 409 (6823), 1007-1011 (2001) MEDLINE 21128732 REFERENCE 2 (residues 1 to 492) AUTHORS Parkhill,J. TITLE Direct Submission JOURNAL Submitted (20-FEB-2001) Submitted on behalf of the Mycobacterium leprae sequencing teams, The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK Unitie de Genetique Moleculaire Bacterienne, Institut Pasteur, 28 rue du Docteur Rouux, 75724, Paris Cedex, France. E-mail: [email protected] COMMENT Notes: Details of M. leprae sequencing at the Sanger Centre are available from http://www.sanger.ac.uk/Projects/M_leprae/ A relational datbase containing the M. leprae sequences is available from http://genolist.pasteur.fr/Leproma/. FEATURES Location/Qualifiers source 1..492 /organism="Mycobacterium leprae" /strain="TN" /db_xref="taxon:1769" Protein 1..492 /product="putative penicillin-binding protein" CDS 1..492 /gene="pbpA" /coded_by="complement(AL583917.1:22189..23667)" /transl_table=11 99
/note="Similar to M. tuberculosis pbpA, penicillin- binding protein, TR:P71586 (EMBL:AL123456) (491 aa); Fasta score E(): 0, 85.7% identity in 490 aa overlap. Also similar to Bacillus subtilis penicillin-binding protein 3, pbpC, SW:PBPC_BACSU (P42971) (668 aa); Fasta score E(): 2.8e- 15, 24.5% identity in 444 aa overlap. Previously sequenced as TR:Q50185 (EMBL:Z70722) (474 aa); Fasta score E(): 0, 96.4% identity in 472 aa overlap. Contains a probable N-terminal signal sequence. Contains Pfam match to entry PF00905 Transpeptidase, Penicillin binding protein transpeptidase domain.~Similar to region of ML1577" ORIGIN 1 mntslrrisv tvmalivlll lnatvtqvft adglradprn qrvlldeysr qrgqitasgq 61 llaysvatdn rfrflrvypn pavyapitgf yslrysstgl eqaedallng sderlfgrrl 121 adfftgrdpr ggnvdttinp rvqqtgwdam qqgcggspck gavvalepst gkilamvstp 181 sydpnllash npeeqaqawr rlhddpnspl inraisetyp pgstfkvitt taalqagatt 241 sdqltaepti plpgstatle nyggvscgpg ptvslsqafa mscntafvql glligadalr 301 smahsfglds npnviplqva estvgvipda aalgmssigq rdvaltplqn aqiaatiana 361 gvtmqpylid nlkgpdlani rtttsyqqhr avspqiaatl telmvgaekv tqqkgaipgv 421 qiasktgtae hgtdprhtpp hawyiafapv qapkvavavl vekgadslfa tggalaapig 481 ravieaalqg ga
100
ponA
Gene name: Class: Start/End: Length: Molecular weight: ponA Rv0050 II.C.3 53663 / 55696 2034 bp / 678 aa 71151 Da Product: penicillin-bonding protein
Description:
Rv0050, (MTCY21D4.13), len: 678, Probable PENICILLIN-BINDING PROTEIN, ponA homologue, highly similar to many, e.g. M. leprae MSGDNAB_6 Mycobacterium leprae cosmid L222 D (686 aa), 82.3% identity in 679 aa overlap.
Translation: MVILLPMVTF TMAYLIVDVP KPGDIRTNQV STILASDGSE IAKIVPPEGN RVDVNLSQVP MHVRQAVIAA EDRNFYSNPG FSFTGFARAV KNNLFGGDLQ GGSTITQQYV KNALVGSAQH GWSGLMRKAK ELVIATKMSG EWSKDDVLQA YLNIIYFGRG AYGISAASKA YFDKPVEQLT VAEGALLAAL IRRPSTLDPA VDPEGAHARW NWVLDGMVET KALSPNDRAA QVFPETVPPD LARAENQTKG PNGLIERQVT RELLELFNID EQTLNTQGLV VTTTIDPQAQ RAAEKAVAKY LDGQDPDMRA AVVSIDPHNG AVRAYYGGDN ANGFDFAQAG LQTGSSFKVF ALVAALEQGI GLGYQVDSSP LTVDGIKITN VEGEGCGTCN IAEALKMSLN TSYYRLMLKL NGGPQAVADA AHQAGIASSF PGVAHTLSED GKGGPPNNGI VLGQYQTRVI DMASAYATLA ASGIYHPPHF VQKVVSANGQ VLFDASTADN TGDQRIPKAV ADNVTAAMEP IAGYSRGHNL AGGRDSAAKT GTTQFGDTTA NKDAWMVGYT PSLSTAVWVG TVKGDEPLVT ASGAAIYGSG LPSDIWKATM DGALKGTSNE TFPKPTEVGG YAGVPPPPPP PEVPPSETVI QPTVEIAPGI TIPIGPPTTI TLAPPPPAPP AATPTPPP Pfam domains present: Not available Prosite patterns present: None
101
LOCUS T10011 474 aa BCT 16-JUL-1999 DEFINITION probable penicillin-binding protein - Mycobacterium leprae. ACCESSION T10011 PID g7476086 VERSION T10011 GI:7476086 DBSOURCE pir: locus T10011; summary: #length 474 #molecular-weight 50415 #checksum 7635; genetic: #gene pbpA; PIR dates: 16-Jul-1999 #sequence_revision 16-Jul-1999 #text_change 16-Jul-1999. KEYWORDS . SOURCE Mycobacterium leprae. ORGANISM Mycobacterium leprae Bacteria; Firmicutes; Actinobacteria; Actinobacteridae; Actinomycetales; Corynebacterineae; Mycobacteriaceae; Mycobacterium. REFERENCE 1 (residues 1 to 474) AUTHORS Cole, S.T. TITLE Direct Submission JOURNAL Submitted (??-AUG-1997) to the EMBL Data Library FEATURES Location/Qualifiers source 1..474 /organism="Mycobacterium leprae" /db_xref="taxon:1769" Protein 1..474 /product="probable penicillin-binding protein" ORIGIN 1 mntslrrisv tvmalivlll lnatvtqvft adglradprn qrvlldeysr qrgqitasgq 61 llaysvatdn rfrflrvypn pavyapitgf yslrysstgl eqaedallng sderlfgrrl 121 adfftgrdpr ggnvdttinp rvqqtgwdam qqgcggspck gavvalepst gkilamvstp 181 sydpnllash npeeqaqawr rlhddpnspl inraisetyp pgstfkvitt taalqagatt 241 sdqltaepti plpgstatle nyggvscdpg ptvslsqhsp crvtprsssw acfigadalr 301 smahsfglds npnviplqva estvgvipda aalgmssigq rdvaltplqn aqiaatiana 361 gvtmqpylid nlkgpdlani rtttsyqqhr avspqiaatl telmvgaekv tqqkgaipgv 421 qiasktgtae hgtdprhtpp hawyiafapv qapkvavavl vekgadslfa tgvr
102
pbpA
Gene name: Class: Start/End: Length: Molecular weight: pbpA Rv0016c II.C.3 18762 / 20234 1473 bp / 491 aa 51576 Da Product: penicillin-binding protein
Description:
Rv0016c, (MTCY10H4.16c), pbpA homologue, len: 491, highly similar to M. leprae, penicillin binding protein, pbpA MLCB1770_11, (474 aa), FASTA scores: opt: 2516 z-score: 2600.4 E(): 0; 82.4% identity in 472 aa overlap. Also similar to E235825, penicillin binding protein, pbpA (325 aa), fasta scores, opt: 1618, E(): 0, (78.3% identity in 323 aa overlap). Also similar to M. tuberculosis MTCY270_5 and MTV003_8;
Translation: MNASLRRISV TVMALIVLLL LNATMTQVFT ADGLRADPRN QRVLLDEYSR QRGQITAGGQ LLAYSVATDG RFRFLRVYPN PEVYAPVTGF YSLRYSSTAL ERAEDPILNG SDRRLFGRRL ADFFTGRDPR GGNVDTTINP RIQQAGWDAM QQGCYGPCKG AVVALEPSTG KILALVSSPS YDPNLLASHN PEVQAQAWQR LGDNPASPLT NRAISETYPP GSTFKVITTA AALAAGATET EQLTAAPTIP LPGSTAQLEN YGGAPCGDEP TVSLREAFVK SCNTAFVQLG IRTGADALRS MARAFGLDSP PRPTPLQVAE STVGPIPDSA ALGMTSIGQK DVALTPLANA EIAATIANGG ITMRPYLVGS LKGPDLANIS TTVGYQQRRA VSPQVAAKLT ELMVGAEKVA QQKGAIPGVQ IASKTGTAEH GTDPRHTPPH AWYIAFAPAQ APKVAVAVLV ENGADRLSAT GGALAAPIGR AVIEAALQGE P Pfam domains present: Not available Prosite patterns present: None