Bacterial Cell Pellets Research Articles

Comparative analysis of vaginal microbiota sampling using menstrual cups and high vaginal swabs in pregnant women living with HIV-1 infection.

Menstrual cups (MCs) are increasingly used to collect cervicovaginal secretions to characterise vaginal mucosal immunology, in conjunction with high vaginal swabs (HVS) for metataxonomics, particularly in HIV transmission studies. We hypothesised that both methods of collecting bacterial biomass are equivalent for 16S rRNA gene sequencing. Cervicovaginal fluid (CVF) samples from 16 pregnant women with HIV-1 (PWWH) were included to represent the major vaginal bacterial community state types (CST I-V). Women underwent sampling during the second trimester by liquid amies HVS followed by a MC (Soft disc™) and samples were stored at -80°C. Bacterial cell pellets obtained from swab elution and MC (500 µL, 1 in 10 dilution) were resuspended in 120 µL PBS for DNA extraction. Bacterial 16S rRNA gene sequencing was performed using V1-V2 primers and were analysed using MOTHUR. Paired total DNA, bacterial load, amplicon read counts, diversity matrices and bacterial taxa were compared by sampling method using MicrobiomeAnalyst, SPSS and R. The total DNA eluted from one aliquot of diluted CVF from an MC was similar to that of a HVS (993ng and 609ng, p=0.18); the mean bacterial loads were also comparable for both methods (MC: 8.0 log10 16S rRNA gene copies versus HVS: 7.9 log10 16S rRNA gene copies, p=0.27). The mean number of sequence reads generated from MC samples was lower than from HVS (MC: 12730; HVS:14830, p=0.05). The α-diversity metrices were similar for both techniques; MC Species Observed: 41 (range 12-96) versus HVS: 47 (range 16-96), p=0.15; MC Inverse Simpson Index: 1.98 (range 1.0-4.0) versus HVS: 0.48 (range 1.0-4.4), p=0.22). The three most abundant species observed were: Lactobacillus iners, Lactobacillus crispatus and Gardnerella vaginalis. Hierarchical clustering of relative abundance data showed that samples obtained using different techniques in an individual clustered in the same CST group. These data demonstrate that despite sampling slightly different areas of the lower genital tract, there was no difference in bacterial load or composition between methods. Both are suitable for characterisation of vaginal microbiota in PWWH. The MC offers advantages, including a higher volume of sample available for DNA extraction and complimentary assays.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Gram-negative bacteria act as a reservoir for aminoglycoside antibiotics that interact with host factors to enhance bacterial killing in a mouse model of pneumonia.

In vitro exposure of multiple Gram-negative bacteria to an aminoglycoside (AG) antibiotic has previously been demonstrated to result in bacterial alterations that interact with host factors to suppress Gram-negative pneumonia. However, the mechanisms resulting in suppression are not known. Here, the hypothesis that Gram-negative bacteria bind and retain AGs, which are introduced into the lung and interact with host defenses to affect bacterial killing, was tested. Following in vitro exposure of one of several, pathogenic Gram-negative bacteria to the AG antibiotics kanamycin or gentamicin, AGs were detected in bacterial cell pellets (up to 208 μg/mL). Using inhibitors of AG binding and internalization, the bacterial outer membrane was implicated as the predominant kanamycin and gentamicin reservoir. Following intranasal administration of gentamicin-bound bacteria or gentamicin solution at the time of infection with live, AG-naïve bacteria, gentamicin was detected in the lungs of infected mice (up to 8 μg/g). Co-inoculation with gentamicin-bound bacteria resulted in killing of AG-naïve bacteria by up to 3-log10, mirroring the effects of intranasal gentamicin treatment. In vitro killing of AG-naïve bacteria mediated by kanamycin-bound bacteria required the presence of detergents or pulmonary surfactant, suggesting that increased bacterial killing inside the murine lung is facilitated by the detergent component of pulmonary surfactant. These findings demonstrate that Gram-negative bacteria bind and retain AGs that can interact with host-derived pulmonary surfactant to enhance bacterial killing in the lung. This may help explain why AGs appear to have unique efficacy in the lung and might expand their clinical utility.

Characterization of Extracellular Biosurfactants Expressed by a Pseudomonas putida Strain Isolated from the Interior of Healthy Roots from Sida hermaphrodita Grown in a Heavy Metal Contaminated Soil.

Pseudomonas putida E41 isolated from root interior of Sida hermaphrodita (grown on a field contaminated with heavy metals) showed high biosurfactant activity. In this paper, we describe data from mass spectrometry and genome analysis, to improve our understanding on the phenotypic properties of the strain. Supernatant derived from P. putida E41 liquid culture exhibited a strong decrease in the surface tension accompanied by the ability for emulsion stabilization. We identified extracellular lipopeptides, putisolvin I and II expression but did not detect rhamnolipids. Their presence was confirmed by matrix-assisted laser desorption and ionization (MALDI) TOF/TOF technique. Moreover, ten phospholipids (mainly phosphatidylethanolamines PE 33:1 and PE 32:1) which were excreted by vesicles were also detected. In contrast the bacterial cell pellet was dominated by phosphatidylglycerols (PGs), which were almost absent in the supernatant. It seems that the composition of extracellular (secreted to the environment) and cellular lipids in this strain differs. Long-read sequencing and complete genome reconstruction allowed the identification of a complete putisolvin biosynthesis pathway. In the genome of P. putida E41 were also found all genes involved in glycerophospholipid biosynthesis, and they are likely responsible for the production of detected phospholipids. Overall this is the first report describing the expression of extracellular lipopeptides (identified as putisolvins) and phospholipids by a P. putida strain, which might be explained by the need to adapt to the highly contaminated environment.

Changes of microbial cell survival, metabolic activity, efflux capacity, and quorum sensing ability of Aggregatibacter actinomycetemcomitans due to antimicrobial photodynamic therapy-induced bystander effects.

The bystander effects, whereby naive (bystander) microbial cells near microbial cells directly exposed to certain treatment show responses that would not have happened in the absence of the directly targeted microbial cells, is recently documented in the field of microbiology. In this article, we discuss that substantial bystander responses are also observed after antimicrobial photodynamic therapy (aPDT) using curcumin (Cur). Bystander effects induced by whole bacterial cell suspension (WBCST), cell-free supernatants fluid (CFSFT), and bacterial cell pellet (BCPT) obtained from A. actinomycetemcomitans culture treated with Cur-aPDT on cell survival, quorum sensing (QS) ability, metabolic activity and efflux capacity of A. actinomycetemcomitans were determined using microbial viability assay, Escherichia coli-based bioassay, XTT reduction method, and ethidium bromide (EtBr) accumulation assay, respectively. A. actinomycetemcomitans cell survival reduced by 82.7% (P = 0.001) and 76.2% (P = 0.01) after exposure to WBCST and CFSFT, respectively. The A. actinomycetemcomitans population increased by 5.5% (P = 0.7) after exposure to BCPT. Bacterial metabolic activity decreased by 42.6% (P = 0.02), 35.3% (P = 0.03), and 9.4% (P = 0.5) after exposure to WBCST, CFSFT, and BCPT, respectively. A. actinomycetemcomitans exposed to WBCST, CFSFT, and BCPT showed a reduction of 83.2% (P = 0.001), 77.2% (P = 0.01) and 21.9% (P = 0.09) in the QS mediator compared to the WBCSU, CFSFU, and BCPU of untreated A. actinomycetemcomitans, respectively. No significant change of the EtBr accumulation was observed in the three preparations of the Cur-aPDT-treated culture (i.e. WBCST, CFSFT, and BCPT) compared to their respective controls. The results of the current study revealed that Cur-aPDT could significantly reduce microbial cell survival, cell metabolic activity, efflux capacity, and QS ability through the bystander effects. As a result, the bystander effects of Cur-aPDT along with the direct effect of Cur-aPDT can enhance the efficiency of aPDT as an adjunct therapeutic strategy for treatment of local infections.

Stability of nitroxide biradical TOTAPOL in biological samples.

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Multicentre randomised controlled trial to investigate usefulness of the rapid diagnostic βLACTA test performed directly on bacterial cell pellets from respiratory, urinary or blood samples for the early de-escalation of carbapenems in septic intensive care unit patients: the BLUE-CarbA protocol

IntroductionThe dramatic increase of the incidence of infections caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) has led to an increase of 50% of carbapenem consumption all around Europe in only 5...

Evaluation of Methods for Inoculating Dry Powder Foods with Salmonella enterica, Enterococcus faecium, or Cronobacter sakazakii

Salmonella and Cronobacter are two bacteria of concern in powdered food ingredients with low water activity, due to their ability to remain viable for long periods of time. There is great interest in studying the survival of these bacteria in powdered foods, but discrepancies have been reported between broth-grown and lawn-grown bacterial cells and their thermal resistance and desiccation tolerance once inoculated onto powdered foods. The purpose of this study was to evaluate three different powdered food inoculation methods, two broth-grown and one lawn-grown. To evaluate these methods on three types of powdered food matrices, Salmonella enterica serovar Typhimurium LT2 (ATCC 700720), Salmonella surrogate Enterococcus faecium (NRRL B-2354), and Cronobacter sakazakii (ATCC 29544) were inoculated onto nonfat dry milk powder, organic soy flour, and all-purpose flour using one of the three previously developed inoculation methods. In the first broth-grown method, labeled broth-grown pelletized inoculation, a bacterial cell pellet was added to powdered foods directly and mixed with a sterile wooden stick. The second broth-grown method, labeled broth-grown spray inoculation, used a chromatography reagent sprayer to spray the bacterial cell suspension onto the powdered foods. The third inoculation method, lawn-grown liquid inoculation, made use of a spot inoculation and a stomacher to incorporate each bacterium into the powdered foods. Results indicated that the method of inoculation of each powder impacted repeatability and bacteria survivability postequilibration (4 to 6 days). Broth-grown spray inoculation, regardless of the powder and bacterium, resulted in the highest log reduction, with an average ∼1-log CFU/g reduction following equilibration. Broth-grown pelletized inoculation resulted in the second-highest log reduction (∼0.79 log CFU/g), and finally, lawn-grown liquid inoculation was the most stable inoculation method of the three, with ∼0.52-log CFU/g reduction. Overall, the results from this inoculation study demonstrate that inoculation methodologies impact the desiccation tolerance and homogeneity of C. sakazakii, E. faecium, and Salmonella Typhimurium LT2.

Antiviral potentials of Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae against selected Enterovirus.

Enteroviruses have been associated with a host of clinical presentations including acute flaccid paralysis (AFP). The site of primary replication for most enteroviruses is the gastrointestinal tract (GIT) and lactic acid bacteria (LAB) may confer protection in the GIT against them. This study therefore investigates the antiviral potential of some selected lactic acid bacteria against enterovirus isolates recovered from AFP cases. The antiviral activities of Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae in broth culture, their cell-free supernatant (CFS), and bacterial cell pellets were assayed against Echovirus 7 (E7), E13, and E19 in a pre- and post-treatment approach using cytopathic effect (CPE) and cell viability (MTT) assay. The tested Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae strains have good antiviral properties against E7 and E19 but not against E13. Lactobacillus amylovorus AA099 shows the highest activity against E19. The pre-treatment approach displays better antiviral activities compared to post-treatment approach. The LAB in broth suspension have better antiviral activities than their corresponding CFS and bacterial pellet. Lactic acid bacteria used in this study have the potential as antiviral agents.

Vaginal microbiome modulates topical antiretroviral drug pharmacokinetics.

Tenofovir gel and dapivirine ring provided variable HIV protection in clinical trials, reflecting poor adherence and possibly biological factors. We hypothesized that vaginal microbiota modulates pharmacokinetics and tested the effects of pH, individual bacteria, and vaginal swabs from women on pharmacokinetics and antiviral activity. Tenofovir, but not dapivirine, uptake by human cells was reduced as pH increased. Lactobacillus crispatus actively transported tenofovir leading to a loss in drug bioavailability and culture supernatants from Gardnerella vaginalis, but not Atopobium vaginae, blocked tenofovir endocytosis. The inhibition of endocytosis mapped to adenine. Adenine increased from 65.5 μM in broth to 246 μM in Gardnerella, but decreased to 9.5 μM in Atopobium supernatants. This translated into a decrease in anti-HIV activity when Gardnerella supernatants or adenine were added to cultures. Dapivirine was also impacted by microbiota, as drug bound irreversibly to bacteria, resulting in decreased antiviral activity. When drugs were incubated with vaginal swabs, 30.7% ± 5.7% of dapivirine and 63.9% ± 8.8% of tenofovir were recovered in supernatants after centrifugation of the bacterial cell pellet. In contrast, no impact of microbiota on the pharmacokinetics of the prodrugs, tenofovir disoproxil fumarate or tenofovir alafenamide, was observed. Together, these results demonstrate that microbiota may impact pharmacokinetics and contribute to inconsistent efficacy.

Inhibition of quorum sensing-mediated virulence in Serratia marcescens by Bacillus subtilis R-18

Serratia marcescens is an opportunistic human pathogen causing various nosocomial infections, most importantly urinary tract infections (UTIs). It exhibits increased resistance towards the conventional antibiotics. This study was aimed to evaluate the anti-virulence effect of a rhizosphere soil bacterium Bacillus subtilis strain R-18 against the uropathogen S. marcescens. First, the bacterial cell-free culture supernatant (CFCS) of B. subtilis strain R-18 was evaluated for its quorum sensing inhibitory (QSI) potential against biomarker strain Chromobacterium violaceum and the test pathogen S. marcescens. The B. subtilis R-18 CFCS effectively inhibited the quorum sensing (QS)-mediated violacein pigment production in C. violaceum and prodigiosin pigment production in S. marcescens. Furthermore, B. subtilis R-18 CFCS was successively extracted with different solvent systems. Of these solvents, B. subtilis R-18 petroleum ether (PE) extract showed inhibition in biofilm formation, protease, lipase, and hemolysin productions in S. marcescens. Fourier transform infrared spectroscopic (FT-IR) analysis revealed the alterations in the cellular components of bacterial cell pellets obtained from B. subtilis R-18 PE extract treated and untreated S. marcescens. The differential gene expression study further validated the downregulation of virulence-associated genes. Characterization of the active principle in B. subtilis R-18 PE extract by gas chromatography–mass spectrometry (GC-MS) analysis showed the presence of multiple compounds with therapeutic values, which could possibly reduce the QS-dependent phenotypes in S. marcescens.

Epithelial cell lysates induce ExoS expression and secretion by Pseudomonas aeruginosa.

The type three secretion system (T3SS) is important for the intracellular survival of Pseudomonas aeruginosa. Known T3SS inducers include low Ca2+, serum or host cell contact. Here, we used corneal epithelial cell lysates to test if host cytosolic factors could also induce the T3SS. Invasive P. aeruginosa strain PAO1 was exposed to cell lysates for 16 h, and expression of T3SS effectors determined by q-PCR and Western immunoblot. Lysate exposure reduced PAO1 growth (∼5-fold) versus trypticase soy broth (TSB), but also resulted in appearance of a protein in culture supernatants, but not bacterial cell pellets, which reacted with antibody raised against ExoS. T3SS-inducing media (TSBi) caused the expression and secretion of ExoS and ExoT. Heat-treated lysates induced the protein; 1:3 diluted lysates did not. The protein that bound anti-ExoS antibody was found in supernatants of lysate-exposed exoT mutants, but not exoS or pscC mutants, suggesting a secreted form of ExoS, albeit slightly larger than that induced by TSBi. Lysate-exposed strain PAK expressed the same protein. Lysates caused PAO1 exoS and exoT gene expression, but only ∼20% and ∼6% of TSBi, respectively. T3SS induction by epithelial cell lysates could help explain T3SS expression by internalized P. aeruginosa.

Petrobactin Is Exported from Bacillus anthracis by the RND-Type Exporter ApeX.

ABSTRACTBacillus anthracis—a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen.

Analysis and optimization of process parameters for production of polyhydroxyalkanoates along with wastewater treatment by Serratia sp. ISTVKR1

A previously reported biodegrading bacterial strain Serratia sp. ISTVKR1 was studied for polyhydroxyalkanoate (PHA) production along with wastewater contaminant removal. Nile red fluorescence, GC–MS, FT-IR, NMR and TEM confirmed the accumulation of homopolymer poly-3-hydroxyvalerate (PHV) within the bacterial cells. Analysis of culture after 72h of bacterial treatment showed maximum COD removal (8.4-fold), non-detection of organic contaminants such as 1H-Cyclopropa [a] naphthalene (R.T.=10.12) using GC–MS and increased proportion of elements like Cr, Mn, Fe, Ni, Cu, Cd and Pb in the bacterial cell pellets by SEM-EDX analysis. Optimization of process parameters for enhanced PHA production along with wastewater treatment done using Response Surface Methodology (RSM) showed 5% and 0.74% increase in the PHA production (0.3368±0.13gL−1) and % COD reduction (88.93±2.41) of wastewater, respectively. The study, thus established the production of PHA along with wastewater contaminant removal by Serratia sp. ISTVKR1.

Analysis of Bacterial Cell Surface Chemical Composition Using Cryogenic X-Ray Photoelectron Spectroscopy.

This chapter describes a method for measuring the average surface chemical composition with respect to lipids, polysaccharides, and peptides (protein + peptidoglycan) for the outer part of the bacterial cell wall. Bacterial cultures grown over night are washed with a buffer or saline at controlled pH. The analysis is done on fast-frozen bacterial cell pellets obtained after centrifugation, and the analysis requires access to X-ray photoelectron spectroscopy instrumentation that can perform analyses at cryogenic temperatures (for example using liquid nitrogen). The method can be used to monitor changes in the cell wall composition following environmental stimuli or genetic mutations. The data obtained originate from the outermost part of the cell wall. Thus, it is expected that for gram-negative bacteria only the outer membrane and part of the periplasmic peptidoglycan layer is probed during analysis, and for gram-positive bacteria only the top nanometers of the peptidoglycan layer of the cell wall is monitored.

Screening and development of DNA aptamers specific to several oral pathogens.

Aptamers are composed of single-stranded oilgonucleotides that can selectively bind desired molecules. It has been reported that RNA or DNA could act as not only a genetic messenger but also a catalyst in metabolic pathways. RNA aptamers (average sizes 40-50 bp) are smaller than antibodies and have strong binding capacities to target molecules, similar to antigen-antibody interactions. Once an aptamer was selected, it can be readily produced in large quantities at low cost. The objectives of this study are to screen and develop aptamers specific to oral pathogens such as Porphyromonas gingivalis, Treponema denticola, and Streptococcus mutans. The bacterial cell pellet was fixed with formaldehyde as a target molecule for the screening of aptamers. The SELEX method was used for the screening of aptamers and a modified western blot analysis was used to verify their specificities. Through SELEX, 40 kinds of aptamers were selected and the specificity of the aptamers to the bacterial cells was confirmed by modified western blot analysis. Through the SELEX method, 40 aptamers that specifically bind to oral pathogens were screened and isolated. The aptamers showed possibility as effective candidates for the detection agents of oral infections.

Biosynthesis of Amphi-enterobactin Siderophores by Vibrio harveyi BAA-1116: Identification of a Bifunctional Nonribosomal Peptide Synthetase Condensation Domain

The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.

Molecular Recognition of the Tes LIM2–3 Domains by the Actin-related Protein Arp7A

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized “orphan” Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1–65Arp7A·LIM2–3Tes·EVH1Mena complex reveals that residues 28–49 of Arp7A contact the LIM2–3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2–3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.