Acquisition of Gonococcal AniA-NorB Pathway by the Neisseria meningitidis Urethritis Clade Confers Denitrifying and Microaerobic Respiration Advantages for Urogenital Adaptation.

Neisseria meningitidis historically has been an infrequent and sporadic cause of urethritis and other urogenital infections. However, a nonencapsulated meningococcal clade belonging to the hyperinvasive clonal complex 11.2 lineage has recently emerged and caused clusters of urethritis cases in the United States and other countries. One of the genetic signatures of the emerging N. meningitidis urethritis clade (NmUC) is a chromosomal gene conversion event resulting in the acquisition of the Neisseria gonorrhoeae denitrification apparatus-the N. gonorrhoeae alleles encoding the nitrite reductase AniA, the nitric oxide (NO) reductase NorB, and the intergenic promoter region. The biological importance of the N. gonorrhoeae AniA-NorB for adaptation of the NmUC to a new environmental niche is investigated herein. We found that oxygen consumption, nitrite utilization, and NO production were significantly altered by the conversion event, resulting in different denitrifying aerobic and microaerobic growth of the clade. Further, transcription of aniA and norB in NmUC isolates differed from canonical N. meningitidis, and important polymorphisms within the intergenic region, which influenced aniA promoter activity of the NmUC, were identified. The contributions of three known meningococcal regulators (NsrR, FNR, and NarQP) in controlling the denitrification pathway and endogenous NO metabolism were distinct. Overall, transcription of aniA was dampened relative to canonical N. meningitidis, and this correlated with the lower NO accumulation in the clade. Denitrification and microaerobic respiration were bolstered, and protection against host-derived NO was likely enhanced. The acquisition of the N. gonorrhoeae denitrification pathway by the NmUC supports the clade's adaptation and survival in a microaerobic urogenital environment.

Probe-based qPCR assay enables the rapid and specific detection of bacterial degrading genes for the pesticide metaldehyde in soil.

Metaldehyde, a molluscicide pesticide, has been identified as a pollutant of concern due to its repeated detection in drinking water, thereby generating numerous compliance failures for water utilities. Biological degradation potential for metaldehyde is widespread in soils, occurring at different rates, but to date, no molecular methods for its assessment have been reported. Here, three genes belonging to a shared metaldehyde-degrading gene cluster present in bacteria were used as candidates for development of a quantitative PCR (qPCR) assay for assessing the metaldehyde-degrading potential in soil. Screening of gene targets, primer pairs and optimization of reaction conditions led to the development of a sensitive and specific probe-based qPCR method for quantifying the mahY metaldehyde-degrading gene from soil. The technique was tested across 8 soils with different compositions and origins. The degrading pathway was detected in 4/8 soils, in which a higher number of gene copies correlated with periods of greater metaldehyde removal. Additionally, swift elimination of the pesticide was observed in soils with an elevated initial number of mahY gene copies. The gene cluster was not detected in other soils, even though metaldehyde removal occurred, indicating that other biological degrading pathways are also important in nature. The method described here is the first one available to estimate the microbial metaldehyde degradation potential and activity in soils, and can also be used to detect degrading microorganisms in systems such as sand filters for water purification or to monitor degrading strains in engineered processes.

Long-term fertilization and tillage regimes have limited effects on structuring bacterial and denitrifier communities in a sandy loam UK soil.

Denitrification causes loss of available nitrogen from soil systems, thereby reducing crop productivity and increasing reliance on agrochemicals. The dynamics of denitrification and denitrifying communities are thought to be altered by land management practices, which affect the physicochemical properties of the soil. In this study, we look at the effects of long-term tillage and fertilization regimes on arable soils following 16 years of treatment in a factorial field trial. By studying the bacterial community composition based on 16S rRNA amplicons, absolute bacterial abundance and diversity of denitrification functional genes (nirK, nirS and nosZ), under conditions of minimum/conventional tillage and organic/synthetic mineral fertilizer, we tested how specific land management histories affect the diversity and distribution of both bacteria and denitrification genes. Bacterial and denitrifier communities were largely unaffected by land management history and clustered predominantly by spatial location, indicating that the variability in bacterial community composition in these arable soils is governed by innate environmental differences and Euclidean distance rather than agricultural management intervention.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

The basis for non-canonical ROK family function in the N-acetylmannosamine kinase from the pathogen Staphylococcus aureus.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

The fitness challenge of studying molecular adaptation.

Advances in bioinformatics and high-throughput genetic analysis increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the molecular-level changes - i.e. the molecular adaptation - that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the molecular level. In particular, this work has examined how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing molecular adaptations is identifying systems and designing experiments that integrate changes in the genome, protein chemistry (molecular phenotype), and fitness. Knowledge of the molecular changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the molecular-level adaptations that connect genetic changes and fitness.

A biotin-conjugated photo-activated CO-releasing molecule (biotinCORM): efficient CO-release from an avidin-biotinCORM protein adduct.

Biotinylated pharmaceuticals are of great interest due to the strong interactions between biotinyl-functionality and streptavidin/avidin, which opens up avenues for efficient targeting and localisation. Three new carbon monoxide-releasing molecules (CO-RMs) have been synthesised and characterised using chemical and biological analysis. An alkyne-containing CO-RM 2 was found to be toxic to RAW 264.7 murine macrophages; and thus therapeutically viable CO-RM 1 was employed as the alkyne precursor for [3 + 2] cycloaddition chemistry enabling a new acid-containing CO-RM 4 and biotin-bioconugate-CO-RM (BiotinCORM 5) to be prepared. CO-RM 4 showed significantly improved solubility and BiotinCORM 5 acts as a photo-CO-RM. We have found that an avidin-CORM adduct of 5 is a CO-releasing protein, releasing CO on irradiation with light (400 nm). The avidin-biotinCORM adduct of 5 was found to have a binding energy of 10 kcal mol-1.

Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Escherichia coli Membrane-Bound [NiFe] Hydrogenase-1.

[NiFe] hydrogenases are electrocatalysts that oxidize H2 at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H2-binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2-induced over-oxidation of the [Fe4S3Cys2] proximal cluster possessed by all O2-tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O2-free electrochemical measurements at high potential that indicate the histidine-mediated [Fe4S3Cys2] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe4S4] center.

Isolation and characterization of metaldehyde-degrading bacteria from domestic soils.

Metaldehyde is a common molluscicide, used to control slugs in agriculture and horticulture. It is resistant to breakdown by current water treatment processes, and its accumulation in drinking water sources leads to regular regulatory failures in drinking water quality. To address this problem, we isolated metaldehyde-degrading microbes from domestic soils. Two distinct bacterial isolates were cultured, that were able to grow prototrophically using metaldehyde as sole carbon and energy source. One isolate belonged to the genus Acinetobacter (strain designation E1) and the other isolate belonged to the genus Variovorax (strain designation E3). Acinetobacter E1 was able to degrade metaldehyde to a residual concentration < 1 nM, whereas closely related Acinetobacter strains were completely unable to degrade metaldehyde. Variovorax E3 grew and degraded metaldehyde more slowly than Acinetobacter E1, and residual metaldehyde remained at the end of growth of the Variovorax E3 strain. Biological degradation of metaldehyde using these bacterial strains or approaches that allow in situ amplification of metaldehyde-degrading bacteria may represent a way forward for dealing with metaldehyde contamination in soils and water.

Toxicity of tryptophan manganese(i) carbonyl (Trypto-CORM), against Neisseria gonorrhoeae.

The potential for carbon monoxide-releasing molecules (CO-RMs) as antimicrobials represents an exciting prospective in the fight against antibiotic resistance. Trypto-CORM, a tryptophan-containing manganese(i) carbonyl, is toxic against E. coli following photo-activation. Here, we demonstrate that Trypto-CORM is toxic against Neisseria gonorrhoeae in the absence of photoactivation. Trypto-CORM toxicity was reversed by the high CO affinity globin leg-haemoglobin (Leg-Hb), indicating that the toxicity is due to CO release. Release of CO from Trypto-CORM in the dark was also detected with Leg-Hb (but not myoglobin) in vitro. N. gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs.

Plasma-Generated Poly(allyl alcohol) Antifouling Coatings for Cellular Attachment.

Conformal poly(allyl alcohol) (PAA) coatings were grown on a biomedical grade polyurethane scaffold using pulsed plasma polymerization of the allyl alcohol monomer. The creation of a continuous wave polymer primer layer increases the interfacial adhesion and stability of a subsequent pulsed plasma deposited PAA film. The resulting PAA coatings are strongly hydrophilic and stable following 7 days incubation in biological media. Films prepared through this energy-efficient, two-step process promote human dermal fibroblast cell culture, while resisting E. coli biofilm formation.

Meningitis in adolescents: the role of commensal microbiota.

The pathogen Neisseria meningitidis causes disease amongst infants and adolescents/young adults. Here we argue that disease amongst adolescents is due largely to interaction between N. meningitidis and other members of the upper respiratory tract microbiota, through a metabolic interaction involving exchange of propionic acid.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR.

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Confocal and fluorescence lifetime imaging sheds light on the fate of a pyrene-tagged carbon monoxide-releasing Fischer carbene chromium complex.

The synthesis of a new pyrene-containing Fischer carbene complex is described. The complex has a broad absorbance spectrum between 300 and 400 nm and, on excitation at 345 nm in CH2Cl2 solution, emission is observed at 395 and 415 nm. Emission is also observed in PBS buffer, but in this case the resulting spectra are much broader. Confocal and fluorescence lifetime imaging indicate that emission occurs on treating HeLa cells with the complex and co-localisation studies demonstrate that this is from the mitochondria and lipid-rich regions of the cell.

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence.

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

A therapeutically viable photo-activated manganese-based CO-releasing molecule (photo-CO-RM).

A new class of photochemically-activated CO-releasing molecule (photo-CO-RM), based on a Mn(CO)(4)(C^N) system, is reported in this study. Three CO molecules are released per CO-RM molecule. Complex 3 is a fast releaser, thermally stable in the dark and a viable therapeutic agent.

Neisseria meningitidis and Neisseria gonorrhoeae are differently adapted in the regulation of denitrification: single nucleotide polymorphisms that enable species-specific tuning of the aerobic-anaerobic switch.

The closely related pathogenic Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. aniA (copper-containing nitrite reductase) is tightly regulated by four transcriptional regulators: FNR (fumarate and nitrate reductase), NarP, FUR (Ferric uptake regulator) and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show in the present study that FUR and NarP are both required for the induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-co-operative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase-binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic-anaerobic switch.

A sweet killer: mesoporous polysaccharide confined silver nanoparticles for antibacterial applications.

Silver nanoparticles (AgNP) confined within porous starch have been prepared in a simple, green and efficient manner, utilising the nanoporous structure of predominantly mesoporous starch (MS) to act as nanoparticle stabiliser, support and reducing surface. MS/AgNP materials present high surface areas (S(BET) > 150 m(2) g(-1)) and mesopore volumes (V(meso) > 0.45 cm(3) g(-1)). The interaction of the AgNP precursor and forming nanoparticle nuclei with the mesoporous domains of the porous polysaccharide, direct porosity to increasingly narrower and more defined pore size distributions, indicative of a degree of cooperative assembly. Transmission electron microscopy images indicated the presence of spherical AgNP of a size reflective of the porous polysaccharide mesopore diameter (e.g., 5-25 nm), whilst XPS analysis confirmed the metallic Ag(0) state. Materials were prepared at relatively low Ag loadings (<0.18 mmol g(-1)), demonstrating excellent antimicrobial activity in solid and liquid phase testing against Gram negative (E. coli) and positive (S. aureus) model bacteria. The resulting materials are biocompatible and present a useful solid porous carbohydrate-based polymer vehicle to control the AgNP size regime and facilitate transference to a biological environment.