The effects of urban green space and road proximity to indoor traffic-related PM2.5, NO2, and BC exposure in inner-city schools.

BACKGROUND: Since there are known adverse health impacts of traffic-related air pollution, while at the same time there are potential health benefits from greenness, it is important to examine more closely the impacts of these factors on indoor air quality in urban schools. OBJECTIVE: This study investigates the association of road proximity and urban greenness to indoor traffic-related fine particulate matter (PM2.5), nitrogen dioxide (NO2), and black carbon (BC) in inner-city schools. METHODS: PM2.5, NO2, and BC were measured indoors at 74 schools and outdoors at a central urban over a 10-year period. Seasonal urban greenness was estimated using the Normalized Difference Vegetation Index (NDVI) with 270 and 1230 m buffers. The associations between indoor traffic-related air pollution and road proximity and greenness were investigated with mixed-effects models. RESULTS: The analysis showed linear decays of indoor traffic-related PM2.5, NO2, and BC by 60%, 35%, and 22%, respectively for schools located at a greater distance from major roads. The results further showed that surrounding school greenness at 270 m buffer was significantly associated (p < 0.05) with lower indoor traffic-related PM2.5: -0.068 (95% CI: -0.124, -0.013), NO2: -0.139 (95% CI: -0.185, -0.092), and BC: -0.060 (95% CI: -0.115, -0.005). These associations were stronger for surrounding greenness at a greater distance from the schools (buffer 1230 m) PM2.5: -0.101 (95% CI: -0.156, -0.046) NO2: -0.122 (95% CI: -0.169, -0.075) BC: -0.080 (95% CI: -0.136, -0.026). These inverse associations were stronger after fully adjusting for regional pollution and meteorological conditions. IMPACT STATEMENT: More than 90% of children under the age of 15 worldwide are exposed to elevated air pollution levels exceeding the WHO's guidelines. The study investigates the impact that urban infrastructure and greenness, in particular green areas and road proximity, have on indoor exposures to traffic-related PM2.5, NO2, and BC in inner-city schools. By examining a 10-year period the study provides insights for air quality management, into how road proximity and greenness at different buffers from the school locations can affect indoor exposure.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Rise and rise of the ABC transporter families.

This review will inevitably be influenced by my personal experience and personal view of the progression of this amazing family of proteins. This has generated a huge literature in over nearly five decades, some ideas have bloomed and faded while others have persisted, other contributions simply become redundant, overtaken by better techniques. At the outset, the pioneers had no idea of the magnitude of the topic they were working on, then a very rough idea of the significance emerged and, progressively, the picture becomes sharper and finally extraordinary. I have tried to produce at least an outline of that progression. My apologies for the also inevitable omissions, especially relating to the mass of biochemical and spectroscopy and genetical studies. I decided to prioritise structural biology because structures when successful are definitive and of course provide a 'visual' image. However, I tried to limit the structural aspects to the proteins that reflected the main advances.

Type I Secretion Systems-One Mechanism for All?

Type I secretion systems (T1SS) are widespread in Gram-negative bacteria, especially in pathogenic bacteria, and they secrete adhesins, iron-scavenger proteins, lipases, proteases, or pore-forming toxins in the unfolded state in one step across two membranes without any periplasmic intermediate into the extracellular space. The substrates of T1SS are in general characterized by a C-terminal secretion sequence and nonapeptide repeats, so-called GG repeats, located N terminal to the secretion sequence. These GG repeats bind Ca2+ ions in the extracellular space, which triggers folding of the entire protein. Here we summarize our current knowledge of how Gram-negative bacteria secrete these substrates, which can possess a molecular mass of up to 1,500 kDa. We also describe recent findings that demonstrate that the absence of periplasmic intermediates, the "classic" mode of action, does not hold true for all T1SS and that we are beginning to realize modifications of a common theme.

Bacillus subtilis Swarmer Cells Lead the Swarm, Multiply, and Generate a Trail of Quiescent Descendants.

Bacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion. IMPORTANCE: Swarming motility enables rapid coordinated surface translocation of a microbial community, preceding the formation of a biofilm. This movement occurs in thin films and involves specialized swarmer cells localized to a narrow zone at the extreme swarm edge. In the B. subtilis system, using a synthetic medium, the swarm front remains as a cellular monolayer for up to 1.5 cm. Swarmers display high-velocity whirls and vortexing and are often assumed to drive community expansion at the expense of cell growth. Surprisingly, little attention has been paid to which cells in a swarm are actually growing and contributing to the overall biomass. Here, we show that swarmers not only lead the population forward but continue to multiply as a source of all cells in the community. We present a model that explains how exponential growth of only a few cells is compatible with the linear expansion rate of the swarm.

Type I Protein Secretion-Deceptively Simple yet with a Wide Range of Mechanistic Variability across the Family.

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process.Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope "tunnel" is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

The Type 1 secretion pathway - the hemolysin system and beyond.

Type 1 secretion systems (T1SS) are wide-spread among Gram-negative bacteria. An important example is the secretion of the hemolytic toxin HlyA from uropathogenic strains. Secretion is achieved in a single step directly from the cytosol to the extracellular space. The translocation machinery is composed of three indispensable membrane proteins, two in the inner membrane, and the third in the outer membrane. The inner membrane proteins belong to the ABC transporter and membrane fusion protein families (MFPs), respectively, while the outer membrane component is a porin-like protein. Assembly of the three proteins is triggered by accumulation of the transport substrate (HlyA) in the cytoplasm, to form a continuous channel from the inner membrane, bridging the periplasm and finally to the exterior. Interestingly, the majority of substrates of T1SS contain all the information necessary for targeting the polypeptide to the translocation channel - a specific sequence at the extreme C-terminus. Here, we summarize our current knowledge of regulation, channel assembly, translocation of substrates, and in the case of the HlyA toxin, its interaction with host membranes. We try to provide a complete picture of structure function of the components of the translocation channel and their interaction with the substrate. Although we will place the emphasis on the paradigm of Type 1 secretion systems, the hemolysin A secretion machinery from E. coli, we also cover as completely as possible current knowledge of other examples of these fascinating translocation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

The mimic chain reaction.

It is sometimes speculated that the equivalent of the polymerase chain reaction might be developed for identification of peptides, proteins or other molecules. In general, though, it is believed that there can be no way to amplify targets such as proteins. Natural amplification systems do, however, exist as in the case of certain autoinducer systems in bacteria. Here, we outline a possible, generic method, the mimic chain reaction, for obtaining peptides with 3-D structures that mimic the 3-D structure of their targets. These targets would include a variety of molecules, including proteins. There are therefore two categories of applications: the ability via amplification firstly to detect a known protein or other target at an extremely low concentration, and secondly to obtain a set of peptides that mimic the structure of an unknown target and that can be used to obtain a 'photofit'.

ABC transporters, mechanisms and biology: an overview.

This chapter concentrates mainly on structural and mechanistic aspects of ABC (ATP-binding cassette) transporters and, as an example of the physiological significance of these proteins, on lipid transport, vitally important for human health. The chapter considers those aspects of ABC transporter function that appear reasonably well established, those that remain controversial and what appear to be emerging themes. Although we have seen dramatic progress in ABC protein studies in the last 20 years, we are still far from a detailed molecular understanding of function. Nevertheless two critical steps - capture and release of allocrites (transport substrates) involving a binding cavity in the membrane domain, and hydrolysis of ATP by the NBD (nucleotide-binding domain) dimer - are now described by persuasive and testable models: alternating access, and sequential firing of catalysis sites respectively. However, these need to be tested rigorously by more structural and biochemical studies. Other aspects considered include the level at which ATP binding and dimer activation are controlled, the nature of the power stroke delivering mechanical energy for transport, and some unexpected and intriguing differences between importers and exporters. The chapter also emphasizes that some ABC transporters, although important for elimination of toxic compounds (xenobiotics), are also increasingly seen to play crucial roles in homoeostatic regulation of membrane biogenesis and function through translocation of endogenous allocrites such as cholesterol. Another emerging theme is the identification of accessory domains and partners for ABC proteins, resulting in a corresponding widening of the range of activities. Finally, what are the prospects for translational research and ABC transporters?

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp+ (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp+ swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

Crystallization and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of the Escherichia coli ABC transporter haemolysin B.

The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.

The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system.

Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

Bacterial metabolic 'toxins': a new mechanism for lactose and food intolerance, and irritable bowel syndrome.

Lactose and food intolerance cause a wide range of gut and systemic symptoms, including gas, gut pain, diarrhoea or constipation, severe headaches, severe fatigue, loss of cognitive functions such as concentration, memory and reasoning, muscle and joint pain, heart palpitations, and a variety of allergies (Matthews and Campbell, 2000; Matthews et al., 2005; Waud et al., 2008). These can be explained by the production of toxic metabolites from gut bacteria, as a result of anaerobic digestion of carbohydrates and other foods, not absorbed in the small intestine. These metabolites include alcohols, diols such as butan 2,3 diol, ketones, acids, and aldehydes such as methylglyoxal (Campbell et al., 2005, 2009). These 'toxins' induce calcium signals in bacteria and affect their growth, thereby acting to modify the balance of microflora in the gut (Campbell et al., 2004, 2007a,b). These bacterial 'toxins' also affect signalling mechanisms in cells around the body, thereby explaining the wide range of symptoms in people with food intolerance. This new mechanism also explains the most common referral to gastroenterologists, irritable bowel syndrome (IBS), and the illness that afflicted Charles Darwin for 50 years (Campbell and Matthews, 2005a,b). We propose it will lead to a new understanding of the molecular mechanism of type 2 diabetes and some cancers.

Importance of eps genes from Bacillus subtilis in biofilm formation and swarming.

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming in Bacillus subtilis strain 168. To assess the role of particular proteins encoded by the group of epsI-epsO genes that form the eps operon, we constructed a series of insertional mutants. The results obtained showed that mutations in epsJ-epsN, but not in the last gene of the eps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of the sfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), the sfp+ strains defective in eps genes (except epsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168 sfp+.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin.

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

The extraordinary diversity of bacterial protein secretion mechanisms.

I have tried to cover the minimal properties of the prolific number of protein secretion systems identified presently, particularly in Gram negative bacteria. New systems, however, are being reported almost by the month and certainly I have missed some. With the accumulating evidence one remains in awe of the complexity of some pathways, with the Type III, IV and VI especially fearsome and impressive. These systems illustrate that protein secretion from bacteria is not only about passage of large polypeptides across a bilayer but also through long tunnels, raising quite different questions concerning mechanisms. The mechanism of transport via the Sec-translocase-translocon is well on the way to full understanding, although a structure of a stuck intermediate would be very helpful. The understanding of the precise details of the mechanism of targeting specificity, and actual polypeptide translocation in other systems is, however, far behind. Groups willing to do the difficult (and risky) work to understand mechanism should therefore be more actively encouraged, perhaps to pursue multidisciplinary, collaborative studies. In writing this review I have become fascinated by the cellular regulatory mechanisms that must be necessary to orchestrate the complex flow of so many polypeptides, targeted by different signals to such a wide variety of transporters. I have tried to raise questions about how this might be managed but much more needs to be done in this area. Clearly, this field is very much alive and the future will be full of revealing and surprising twists in the story.