Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb3 oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host

Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae

Biochemical Characterization of the O-Linked Glycosylation Pathway in Neisseria gonorrhoeae Responsible for Biosynthesis of Protein Glycans Containing N,N '-Diacetylbacillosamine

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607–624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447–4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N′-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.National Institutes of Health (U.S.) (Grant GM039334)Research Council of Norway (Grant 166931)Research Council of Norway (Grant 183613)Research Council of Norway (Grant 183814)National Institutes of Health (U.S.) (Biotechnology Training Program Grant T32-GM08334

Concerted Spatio-Temporal Dynamics of Imported DNA and ComE DNA Uptake Protein during Gonococcal Transformation

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane

Genetic, Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species▿ †

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution

Influence of minor pilin-like proteins on PilE post-translational modifications.

<p>Deconvoluted mass spectra of intact PilE from ESI MS analysis with multiple protein forms representing species differing in glycosylation and phospho-form modifications are shown. Minor peaks and peaks connected with Na⁺ and/or K⁺ adducts in the mass spectra are not marked. PilE was isolated from A) the wild-type (N400) background, B) the <i>pilV</i> mutant (KS790), C) the <i>pilC</i> mutant (KS789), D) the <i>pilH</i> mutant (KSKS801), E) the <i>pilI</i> mutant (KS804), F) the <i>pilJ</i> mutant (KS806), and G) from the <i>pilK</i> mutant (KS810).</p

Detection of acetic acid in the supernatants of an alkaline incubation mixture of Tfp preparations

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p> Total ion chromatograms (TIC) of the supernatant of the alkaline incubation mixtures of PilE at pH 10.5 after 0, 1 and 24 h (insets with selected ion chromatograms (SIC) of 60 for acetic acid). Electron impact ionization (EI) spectrum of the compound eluting at 11.2 min confirms formation of acetic acid over time

-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure-10

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure-4

<p><b>Copyright information:</b></p><p>Taken from "-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure"</p><p></p><p>Molecular Microbiology 2007;65(3):607-624.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC1976384.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p