Structure and function of DsbA, a key bacterial oxidative folding catalyst

Since its discovery in 1991, the bacterial periplasmic oxidative folding catalyst DsbA has been the focus of intense research. Early studies addressed why it is so oxidizing and how it is maintained in its less stable oxidized state. The crystal structure of Escherichia coli DsbA (EcDsbA) revealed that the oxidizing periplasmic enzyme is a distant evolutionary cousin of the reducing cytoplasmic enzyme thioredoxin. Recent significant developments have deepened our understanding of DsbA function, mechanism, and interactions: the structure of the partner membrane protein EcDsbB, including its complex with EcDsbA, proved a landmark in the field. Studies of DsbA machineries from bacteria other than E. coli K-12 have highlighted dramatic differences from the model organism, including a striking divergence in redox parameters and surface features. Several DsbA structures have provided the first clues to its interaction with substrates, and finally, evidence for a central role of DsbA in bacterial virulence has been demonstrated in a range of organisms. Here, we review current knowledge on DsbA, a bacterial periplasmic protein that introduces disulfide bonds into diverse substrate proteins and which may one day be the target of a new class of anti-virulence drugs to treat bacterial infection. Antioxid. Redox Signal. 14, 1729–1760.

DSB proteins and bacterial pathogenicity

If DNA is the information of life, then proteins are the machines of life — but they must be assembled and correctly folded to function. A key step in the protein-folding pathway is the introduction of disulphide bonds between cysteine residues in a process called oxidative protein folding. Many bacteria use an oxidative protein-folding machinery to assemble proteins that are essential for cell integrity and to produce virulence factors. Although our current knowledge of this machinery stems largely from Escherichia coli K-12, this view must now be adjusted to encompass the wider range of disulphide catalytic systems present in bacteria.

Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

Assessment of the short-term success of a translocation of lesser short-tailed bats Mystacina tuberculata

Translocation is a powerful tool that has been used in the conservation of a wide range of taxa. However, few translocations of bats have been attempted and we know of no successes. The few translocations which have been attempted have either failed due to dispersal from the release site or have not been monitored sufficiently to determine the cause of failure. We assessed the short-term success of a translocation of lesser short-tailed bats Mystacina tuberculata by the New Zealand Department of Conservation, where 3 release methods were used to minimise dispersal or mortality: bats were juveniles, were maintained in captivity at the release site, and were provided with supplementary food and roosts following release. Success was assessed by determining if founders remained at the release site and maintained condition (weight). Recapture showed that at least 9 of the 20 bats remained at the release site 232 d after release. There was weak evidence that bats lost weight, although final weights were comparable to those of bats from a natural population. However, all bats caputred 8 mo after release had damaged, infected ears and some were balding. The problem was treated but recurred, and bats were returned to captivity. Our results are th first to demonstrate that translocated bats can remain at their release site and survive. However, disease may be an issue in future translocations.

Translocation of bats as a conservation strategy : previous attempts and potential problems

Translocation is an increasingly popular conservation tool from which a wide range of taxa have benefited. However, to our knowledge, bats have not been translocated successfully. Bats differ behaviourally, morphologically and physiologically from the taxa for which translocation the- ory has been developed, so existing guidelines may not be directly transferable. We review previous translocations of bats and discuss characteristics of bats that may require special consideration dur- ing translocation. Their vagility and homing ability, coloniality, roost requirements, potential ability to transmit diseases, susceptibility to anthropomorphic impacts, and cryptic nature have implications for establishing populations, effects of these populations on the release site, and ability to monitor translocation success following release. We hope that our discussion of potential problems will be able to supplement the existing, more generic guidelines to provide a starting point for the planning of bat translocations.

The potential availability of roosting sites for lesser short-tailed bats (Mystacina tuberculata) on Kapiti Island, New Zealand: Implications for a translocation

Lesser short-tailed bats (Mystacina tuberculata) have recently been translocated to Kapiti Island in an attempt to form a new population of this threatened species. However, the island's vegetation is regenerating, and there was doubt that the forests provided enough large trees with cavities for bats to roost in. This study measured the availability of tree-trunk cavities of the right size for potential roost sites on Kapiti Island, and assessed if habitat restoration would be required to increase the translocation's chance of success. First, trees with cavities accessible to us were sampled in six of Kapiti Island's forest types. Size variables known to affect roost site selection by lesser short-tailed bats at the tree and cavity level were measured. Trees were classified as containing cavities that could potentially provide suitable roosts if their values for all variables measured fell within the range of roosts used by lesser short-tailed bats in natural populations. Roosts were classified as suitably sized for solitary bats or for colonies, using measurements from both types of roosts in natural populations. Second, the density of these potential roost cavities was calculated. Cavities of a size potentially suitable for colonies were found in four of the six forest types at densities ranging from 3.2 +/- 3.2 SE to 52.4 +/- 14.0 trees per ha. Density of potential solitary roosts was much higher. Not all potential cavities will be suitable because they may be damp, poorly insulated, or have an unsuitable microclimate. Nevertheless, our estimates indicated that the two most extensive forest types each contained thousands of potential cavities of a size suitable for colonies of lesser short-tailed bats. In addition, there were tens of thousands of cavities large enough to shelter solitary bats. Roost habitat restoration appears unnecessary to assist translocated Mystacina tuberculata on Kapiti Island.

Evidence of homing following translocation of long‐tailed bats (Chalinolobus tuberculatus) at Grand Canyon Cave, New Zealand

Homing behaviour in the New Zealand long-tailed bat (Chalinolobus tuberculatus), a temperate insectivorous species, was investigated at Grand Canyon Cave, central North Island. A pilot study of nine adult male bats was conducted to determine whether use of the cave was regular enough for a homing study. Eight bats returned to the cave over the 3 week monitoring period, six on the night of the following release. Nine additional bats carrying radio transmitters were then released at three sites (three at each site) c.5, 10 and 20km due east of the border of, and outside the population's known familiar area respectively. All but one of these nine was subsequently detected at the cave. Results suggest that adult long-tailed bats are able to return home following displacement both inside and outside their familiar area. Implications of these findings for translocations of bats and the possessions of a potential long distance navigation system by this species are discussed.

Bacterial adherence and biofilm formation on medical implants : a review

Biofilms are a complex group of microbial cells that adhere to the exopolysaccharide matrix present on the surface of medical devices. Biofilm-associated infections in the medical devices pose a serious problem to the public health and adversely affect the function of the device. Medical implants used in oral and orthopedic surgery are fabricated using alloys such as stainless steel and titanium. The biological behavior, such as osseointegration and its antibacterial activity, essentially depends on both the chemical composition and the morphology of the surface of the device. Surface treatment of medical implants by various physical and chemical techniques are attempted in order to improve their surface properties so as to facilitate bio-integration and prevent bacterial adhesion. The potential source of infection of the surrounding tissue and antimicrobial strategies are from bacteria adherent to or in a biofilm on the implant which should prevent both biofilm formation and tissue colonization. This article provides an overview of bacterial biofilm formation and methods adopted for the inhibition of bacterial adhesion on medical implants

Conformational Spread as a Mechanism for Cooperativity in the Bacterial Flagellar Switch

The bacterial flagellar switch that controls the direction of flagellar rotation during Chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread-the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes.

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

Potentiation of ciprofloxacin action against Gram-negative bacterial biofilms by a nitroxide

We previously showed that soluble nitroxides (nitric oxide analogues) mimicked the well-established ability of nitric oxide to cause biofilm dispersal and further showed that these compounds could prevent biofilm formation. Here, we investigated the effect of the nitroxide carboxy-TEMPO in combination with sub μg/ml concentrations of ciprofloxacin on pre-formed flow cell biofilms formed by Gram-negative bacteria. Combination therapy led to substantial eradication of existing biofilms formed by Pseudomonas aeruginosa PA14 (99.3%) and Escherichia coli O157 (93%).

Identification of bacterial community composition in freshwater aquaculture system farming of Litopenaeus vannamei reveals distinct temperature-driven patterns

Change in temperature is often a major environmental factor in triggering waterborne disease outbreaks. Previous research has revealed temporal and spatial patterns of bacterial population in several aquatic ecosystems. To date, very little information is available on aquaculture environment. Here, we assessed environmental temperature effects on bacterial community composition in freshwater aquaculture system farming of Litopenaeus vannamei (FASFL). Water samples were collected over a one-year period, and aquatic bacteria were characterized by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rDNA pyrosequencing. Resulting DGGE fingerprints revealed a specific and dynamic bacterial population structure with considerable variation over the seasonal change, suggesting that environmental temperature was a key driver of bacterial population in the FASFL. Pyrosequencing data further demonstrated substantial difference in bacterial community composition between the water at higher (WHT) and at lower (WLT) temperatures in the FASFL. Actinobacteria, Proteobacteria and Bacteroidetes were the highest abundant phyla in the FASFL, however, a large number of unclassified bacteria contributed the most to the observed variation in phylogenetic diversity. The WHT harbored remarkably higher diversity and richness in bacterial composition at genus and species levels when compared to the WLT. Some potential pathogenenic species were identified in both WHT and WLT, providing data in support of aquatic animal health management in the aquaculture industry.