Lytic transglycosylases LtgA and LtgD perform distinct roles in remodeling, recycling and releasing peptidoglycan in Neisseria gonorrhoeae.

Neisseria gonorrhoeae releases peptidoglycan (PG) fragments during infection that provoke a large inflammatory response and, in pelvic inflammatory disease, this response leads to the death and sloughing of ciliated cells of the Fallopian tube. We characterized the biochemical functions and localization of two enzymes responsible for the release of proinflammatory PG fragments. The putative lytic transglycosylases LtgA and LtgD were shown to create the 1,6-anhydromuramyl moieties, and both enzymes were able to digest a small, synthetic tetrasaccharide dipeptide PG fragment into the cognate 1,6-anhydromuramyl-containing reaction products. Degradation of tetrasaccharide PG fragments by LtgA is the first demonstration of a family 1 lytic transglycosylase exhibiting this activity. Pulse-chase experiments in gonococci demonstrated that LtgA produces a larger amount of PG fragments than LtgD, and a vast majority of these fragments are recycled. In contrast, LtgD was necessary for wild-type levels of PG precursor incorporation and produced fragments predominantly released from the cell. Additionally, super-resolution microscopy established that LtgA localizes to the septum, whereas LtgD is localized around the cell. This investigation suggests a model where LtgD produces PG monomers in such a way that these fragments are released, whereas LtgA creates fragments that are mostly taken into the cytoplasm for recycling.

Mating pair formation homologue TraG is a variable membrane protein essential for contact-independent type IV secretion of chromosomal DNA by Neisseria gonorrhoeae.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the surrounding milieu. The DNA is effective in transforming gonococci in the population, and this mechanism of DNA donation may contribute to the high degree of genetic diversity in this species. Similar to other F-like T4SSs, the gonococcal T4SS requires a putative membrane protein, TraG, for DNA transfer. In F-plasmid and related systems, the homologous protein acts in pilus production, mating pair stabilization, and entry exclusion. We characterized the localization, membrane topology, and variation of TraG in N. gonorrhoeae. TraG was found to be an inner-membrane protein with one large periplasmic region and one large cytoplasmic region. Each gonococcal strain carried one of three different alleles of traG. Strains that carried the smallest allele of traG were found to lack the peptidoglycanase gene atlA but carried a peptidoglycan endopeptidase gene in place of atlA. The purified endopeptidase degraded gonococcal peptidoglycan in vitro, cutting the peptide cross-links. Although the other two traG alleles functioned for DNA secretion in strain MS11, the smallest traG did not support DNA secretion. Despite the requirement for a mating pair stabilization homologue, static coculture transformation experiments demonstrated that DNA transfer was nuclease sensitive and required active uptake by the recipient, thus demonstrating that transfer occurred by transformation and not conjugation. Together, these results demonstrate the TraG acts in a process of DNA export not specific to conjugation and that different forms of TraG affect what substrates can be transported.

The lytic transglycosylases of Neisseria gonorrhoeae.

Neisseria gonorrhoeae encodes five lytic transglycosylases (LTs) in the core genome, and most gonococcal strains also carry the gonococcal genetic island that encodes one or two additional LTs. These peptidoglycan (PG)-degrading enzymes are required for a number of processes that are either involved in the normal growth of the bacteria or affect the pathogenesis and gene transfer aspects of this species that make N. gonorrhoeae highly inflammatory and highly genetically variable. Systematic mutagenesis determined that two LTs are involved in producing the 1,6-anhydro PG monomers that cause the death of ciliated cells in Fallopian tubes. Here, we review the information available on these enzymes and discuss their roles in bacterial growth, cell separation, autolysis, type IV secretion, and pathogenesis.

Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology.

Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the Δng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.

Recognition of (2S)-aminomalonyl-acyl carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in zwittermicin A biosynthesis.

Polyketide synthases elongate a polyketide backbone by condensing carboxylic acid precursors that are thioesterified to either coenzyme A or an acyl carrier protein (ACP). Two of the three known ACP-linked extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP, are found in the biosynthesis of the agriculturally important antibiotic zwittermicin A. We previously reconstituted the formation of (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP from the primary metabolites l-serine and 1,3-bisphospho-d-glycerate. In this report, we characterize the two acyltransferases involved in the specific transfer of the (2S)-aminomalonyl and (2R)-hydroxymalonyl moieties from the ACPs associated with extender unit formation to the ACPs integrated into the polyketide synthase. This work establishes which acyltransferase recognizes each extender unit and also provides insight into the substrate selectivity of these enzymes. These are important step toward harnessing these rare polyketide synthase extender units for combinatorial biosynthesis.

Biosynthesis of polyketide synthase extender units.

This review covers the biosynthesis of extender units that are utilized for the assembly of polyketides by polyketide synthases. The metabolic origins of each of the currently known polyketide synthase extender units are covered.

Formation and characterization of acyl carrier protein-linked polyketide synthase extender units.

Polyketide natural products are assembled by the condensation of an initiating precursor, or starter unit, with a series of additional precursors referred to as extender units. While there are a number of polyketide synthase starter units, there are currently only seven known polyketide synthase extender units. Polyketide synthase extender units thioesterified to coenzyme A have been known for some time; however, polyketide synthase extender units thioesterified to acyl carrier proteins (ACPs) have been identified only recently. Two of them, (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP, are found in the biosynthetic pathway of the antibiotic zwittermicin A in Bacillus cereus UW85. The focus of this chapter is the in vitro formation of (2R)-hydroxymalonyl-ACP and (2S)-aminomalonyl-ACP and the characterization of these extender units using high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Nonribosomal peptide synthetases involved in the production of medically relevant natural products.

Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units.

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster.

The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.