Negative Control Strain Research Articles

Copper Accumulation Efficiency in Different Recombinant Microorganism Strains Available for Bioremediation of Heavy Metal-Polluted Waters.

The aim of this research was to investigate the bioremediation conditions of copper in synthetic water. In the present study, copper ions accumulation efficiency was determined using various genetically modified strains of Saccharomyces cerevisiae (EBY100, INVSc1, BJ5465, and GRF18), Pichia pastoris (X-33, KM71H), Escherichia coli (XL10 Gold, DH5α, and six types of BL21 (DE3)), and Escherichia coli BL21 (DE3) OverExpress expressing two different peroxidases. Viability tests of yeast and bacterial strains showed that bacteria are viable at copper concentrations up to 2.5 mM and yeasts up to 10 mM. Optical emission spectrometry with inductively coupled plasma analysis showed that the tolerance of bacterial strains on media containing 1 mM copper was lower than the tolerance of yeast strains at the same copper concentration. The E. coli BL21 RIL strain had the best copper accumulation efficiency (4.79 mg/L of culture normalized at an optical density of 1.00), which was 1250 times more efficient than the control strain. The yeast strain S. cerevisiae BJ5465 was the most efficient in copper accumulation out of a total of six yeast strains used, accumulating over 400 times more than the negative control strain. In addition, E. coli cells that internally expressed recombinant peroxidase from Thermobifida fusca were able to accumulate 400-fold more copper than cells that produced periplasmic recombinant peroxidases.

Abstract 576: ACTM-838, a novel immunotherapy that enriches in solid tumors after IV dosing and comprehensively reverses the immunosuppressive TME to promote durable anti-tumor immunity

Abstract ACTM-838 is an avirulent bacterial immunotherapy that encodes an engineered IL-15 (IL-15plex) and a constitutively active STING variant (eSTING). The ACTM-838 strain is a highly modified, attenuated S. Typhimurium that lacks several major inflammatory and immunogenic components on the surface of the microbe and is designed to naturally and specifically enrich in the tumor microenvironment (TME) via auxotrophic dependency on metabolites of the adenosine pathway and purines, to achieve tumor-specific payload delivery. ACTM-838 was selectively internalized by phagocytic, primary human antigen presenting cells (APCs) but not endothelial cells. ACTM-838 was stable in human whole blood and is not inactivated by complement. Human macrophage uptake of labeled ACTM-838 led to acidic lysosomes and nuclear delivery with a concomitant increase in expression of encoded IL-15plex and eSTING payloads such as IL-15, IRF3 reporter activity and IFN ⍺/β secretion, respectively. Primary human and murine macrophages polarized towards a dual co-stimulatory and phagocytic M1/M2 dual phenotype in response to ACTM-838. ACTM-838 delivered eSTING payload in STING knockout macrophages and induced significantly higher Type I IFN production over a negative control strain. IV delivered ACTM-838 showed dose and payload dependent tumor enrichment and potent single-agent efficacy in two independent syngeneic tumor models, EMT6 (orthotopic) and MC38. Sustained memory anti-tumor immunity upon tumor rechallenge in cured animals was demonstrated. ACTM-838 treated tumors exhibited a significant increase in human IL-15plex protein payload. ACTM-838 as a single agent induced profound immune reprogramming and remodeling of the TME over time through increased infiltration and activation of myeloid, NK, B and cytolytic CD8 T cells, MHC class I and II, antigen presentation as well as decreased Treg, PD1+Lag3+ exhausted T cells, and a remodeled stromal signature, as assessed by bulk RNAseq, and confirmed by flow cytometry. Synergistic anti-tumor activity was observed in multiple tumor models when ACTM-838 was dosed in combination with anti-PD1, safely inducing complete responses. No significant body weight loss or death was observed in any treatment regimens of ACTM-838 alone (single or multiple doses) or in combination with anti-PD1. Analyses of public RNAseq datasets from patients with primary or metastatic disease revealed cancer types with high levels of myeloid infiltration and adenosine enrichment, suggesting that ACTM-838 efficacy could provide benefit in these indications. ACTM-838 is a novel immunotherapy delivering IL-15plex + eSTING payloads to phagocytic APCs, inducing a durable anti-tumor immune response, after IV dosing. It possesses a compelling safety profile, and is currently in IND-enabling, preclinical development. Citation Format: Ping Fang, Kyle R. Cron, Oanh Pham, Julie R. Janes, John Brandenburg, Fauna Yarza, Bret Peterson, Christopher Thanos, Chan Whiting, Akshata R. Udyavar. ACTM-838, a novel immunotherapy that enriches in solid tumors after IV dosing and comprehensively reverses the immunosuppressive TME to promote durable anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 576.

Antibiotic resistance and virulence characteristics analysis of a carbapenem-resistant hypervirulent Klebsiella pneumoniae

Objective: To characterize the antibiotic resistance and virulence in a carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A CRKP (designated K. pneumoniae C35) was isolated from a stool sample. The minimal inhibitory concentrations of antimicrobial agents were determined using the broth microdilution method. Whole-genome sequencing and genome analysis were performed to identify the antibiotic resistance and virulence genes. The genetic relationship among K. pneumoniae C35 and other CRKP isolates from our hospital was analyzed by single nucleotide polymorphism (SNP) typing of core genomes. Conjugation experiments were carried out by filter mating to evaluate the transferability and efficiency of resistance genes. The virulence phenotype was determined by Galleria mellonella infection model. Results: K. pneumoniae C35 exhibited resistance to the majority of tested antibiotics, especially carbapenems, sulbactam, and polymyxins. SNP typing showed that K. pneumoniae C35 shared a high degree of sequence homology with several CRKP isolates from different wards. This ST11 CRKP carried 13 resistance genes, including blaKPC-2, blaCTX-M-199, mcr-1, and tet(A) variant. blaKPC-2 gene was located on an IncFⅡ plasmid with>69 800 bp in size, blaCTX-M-199 and mcr-1 genes were located on an IncI2 plasmid (>64 800 bp), and tet(A) variant was located on an unknown Inc-type plasmid (83 628bp). All these three plasmids were conjugative. K. pneumoniae C35 was found to harbor rmpA, rmpA2, and iucABCD aerobactin-related genes, and was considered to be classic carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP). The virulence potential of this strain was confirmed in a Galleria mellonella infection model. The survival rate of the larvae injected with strain C35 at 48 h after infection was significantly lower than that of negative control strain (16.7% vs 80.0%). Conclusion: Multiple conjugative plasmids are identified in a faecal CR-hvKP. The IncI2 plasmid co-carrying both blaCTX-M-199 and mcr-1 genes is firstly identified in CR-hvKP. The emergence of such strain should be alerted and active surveillance is warranted.

Roles of rpoN in biofilm formation of Vibrio alginolyticus HN08155 at different cell densities

RpoN (δ54) as a global regulator controls crucialvirulence-associated phenotype, which can regulate flagellum and exopolysaccharides (EPS) during pathogenic biofilm formation. However, the knowledge of the roles of rpoN in biofilm formation of V. alginolyticus is limited, especially at different cell densities. Herein, deletion mutant strain ΔrpoN, complementary strain ΔrpoN-C and negative control strain ΔrpoN-Z were constructed to investigate the effects of rpoN on biofilm formation of V. alginolyticus HN08155 based on flagellum and EPS at different cell density conditions. The results showed that all of strains can form biofilm, and biofilms of strains with rpoN were formed at low cell density (LCD) and detached at high cell density (HCD), while those of ΔrpoN and ΔrpoN-Z were absent at LCD and accumulated excessively with a spotty pellicle at HCD without detaching. The EPS contents of strains with rpoN was greater than that of ΔrpoN and ΔrpoN-Z at LCD, while the opposite trends were observed at HCD. The expression levels of rpoN were quantified, which were consistent with the trend of biofilm formation. It's worth noting that absence of rpoN resulted in the failure of biofilm detachment, lacking of flagellum and decreasing motility, indicating that rpoN was not necessary for biofilm formation, but it was essential for biofilm detachment.

Direct detection of intact Klebsiella pneumoniae carbapenemase variants from cell lysates: Identification, characterization and clinical implications

IntroductionCarbapenemase-producing organisms (CPOs) are a growing threat to human health. Among the enzymes conferring antibiotic resistance produced by these organisms, Klebsiella pneumoniae carbapenemase (KPC) is considered to be a growing global health threat. Reliable and specific detection of this antibiotic resistance-causing enzyme is critical both for effective therapy and to mitigate further spread. ObjectivesThe objective of this study is to develop an intact protein mass spectrometry-based method for detection and differentiation of clinically-relevant KPC variants directly from bacterial cell lysates. The method should be specific for any variant expressed in multiple bacterial species, limit false positive results and be rapid in nature to directly influence clinical outcomes. MethodsLysates obtained directly from bacterial colonies were used for intact protein detection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bottom-up and top-down proteomic methods were used to characterize the KPC protein targets of interest. Comparisons between KPC-producing and KPC-non-producing isolates from a wide variety of species were also performed. ResultsCharacterization of the mature KPC protein revealed an unexpected signal peptide cleavage site preceding an AXA signal peptide motif, modifying the molecular weight (MW) of the mature protein. Taking the additional AXA residues into account allowed for direct detection of the intact protein using top-down proteomic methods. Further validation was performed by transforming a KPC-harboring plasmid into a negative control strain, followed by MS detection of the KPC variant from the transformed cell line. Application of this approach to clearly identify clinically-relevant variants among several species is presented for KPC-2, KPC-3, KPC-4 and KPC-5. ConclusionDirect detection of these enzymes contributes to the understanding of occurrence and spread of these antibiotic-resistant organisms. The ability to detect intact KPC variants via a simple LC-MS/MS approach could have a direct and positive impact on clinical therapy, by providing both direction for epidemiological tracking and appropriate therapy.

Validating the inhibitory effects of d- and l-serine on the enzyme activity of d-3-phosphoglycerate dehydrogenases that are purified from Pseudomonas aeruginosa, Escherichia coli and human colon

BackgroundWe previously demonstrated that the serA gene is associated with bacterial pathogenicity, including bacterial penetration through the Caco-2 cell monolayers, bacterial motility, bacterial adherence, and fly mortality. l-Serine is known to inhibit the d-3-phosphoglycerate dehydrogenase (PGDH) activity of the SerA protein, and it significantly reduced the bacterial pathogenicity as described above. We also demonstrated that in a PGDH assay using crude extracts isolated from overnight cultures of E. coli overexpressing the P. aeruginosa serA gene, l-serine inhibited the PGDH activity of the SerA protein. The basal PGDH activity of the negative control strain was high, presumably due to contamination of unknown proteins in the crude extracts. Therefore, to further confirm the direct inhibition of PGDH activity of P. aeruginosa SerA by l-serine, we purified and characterized the PGDH from P. aeruginosa and compared it with the previously characterized PGDHs from E. coli, and the human colon as controls.ResultsOptimum pH and ionic strength of the purified PGDHs were different depending on the three species; optimal activity of P. aeruginosa PGDH was at pH 7.5 with 50–100 mM Tris–HCl, E. coli PGDH was at pH 8.5 with 100–200 mM Tris–HCl, and human PGDH was at pH 9.0 with 100–200 mM Tris–HCl. The addition of l-serine reduced the activity of PGDH from P. aeruginosa and E. coli, but not the PGDH from human colon. The median inhibitory concentration (IC50) of l-serine was 630 μM for P. aeruginosa and 250 μM for E. coli, while IC50 of d-serine was much higher than that of l-serine; 76 mM in P. aeruginosa PGDH and 45 mM in E. coli PGDH.ConclusionsThese results suggest that l-serine significantly repressed P. aeruginosa pathogenicity through direct inhibition of the PGDH activity, but was not able to inhibit the human PGDH activity. Oral administration of l-serine to compromised hosts might interfere with bacterial translocation and prevent gut-derived sepsis caused by P. aeruginosa through inhibition of the function of the serA gene product.

Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa

Objective: To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect Pseudomonas aeruginosa in clinic. Methods: (1) The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. (2) One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×10(7,) 1×10(6,) 1×10(5,) 1×10(4,) 1×10(3,) 1×10(2,) and 1×10(1) colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (3) The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains (Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida) were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (4) The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software. Results: (1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. (2) Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×10(1) CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×10(2) CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×10(1)-1×10(7) CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×10(1) CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×10(2) CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×10(3)-1×10(7) CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida. (4) In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%. Conclusions: The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.

Enhanced production of a diastereomer type of mannosylerythritol lipid-B by the basidiomycetous yeast Pseudozyma tsukubaensis expressing lipase genes from Pseudozyma antarctica.

Basidiomycetous yeasts in the genus Pseudozyma are known to produce extracellular glycolipids called mannosylerythritol lipids (MELs). Pseudozyma tsukubaensis produces a large amount of MEL-B using olive oil as the sole carbon source (>70g/L production). The MEL-B produced by P. tsukubaensis is a diastereomer type of MEL-B, which consists of 4-O-β-D-mannopyranosyl-(2R,3S)-erythritol as a sugar moiety, in contrast to the conventional type of MELs produced by P. antarctica, which contain 4-O-β-D mannopyranosyl-(2S,3R)-erythritol. In this study, we attempted to increase the production of the diastereomer type of MEL-B in P. tsukubaensis 1E5 by introducing the genes encoding two lipases, PaLIPAp (PaLIPA) and PaLIPBp (PaLIPB) from P. antarctica T-34. Strain 1E5 expressing PaLIPA exhibited higher lipase activity than the strain possessing an empty vector, which was used as a negative control. Strains of 1E5 expressing PaLIPA or PaLIPB showed 1.9- and 1.6-fold higher MEL-B production than the negative control strain, respectively, and oil consumption was also accelerated by the introduction of these lipase genes. MEL-B production was estimated using time course analysis in the recombinant strains. Strain 1E5 expressing PaLIPA produced 37.0±1.2g/L of MEL-B within 4days of cultivation, whereas the strain expressing an empty vector produced 22.1±7.5g/L in this time. Overexpression of PaLIPA increased MEL-B production by P. tsukubaensis strain 1E5 from olive oil as carbon source by more than 1.7-fold.

Biocontrol Activity of Volatile-Producing Bacillus megaterium and Pseudomonas protegens against Aspergillus flavus and Aflatoxin Production on Stored Rice Grains

In our previous study, three bacterial strains, Bacillus megaterium KU143, Microbacterium testaceum KU313, and Pseudomonas protegens AS15, were selected as effective biocontrol agents against Aspergillus flavus on stored rice grains. In this study, we evaluated the inhibitory effects of the volatiles produced by the strains on A. flavus growth and aflatoxin production on stored rice grains. The three strains significantly reduced mycelial growth of A. flavus in dual-culture assays compared with the negative control strain, Sphingomonas aquatilis KU408, and an untreated control. Of these tested strains, volatiles produced by B. megaterium KU143 and P. protegens AS15 markedly inhibited mycelial growth, sporulation, and conidial germination of A. flavus on agar medium and suppressed the fungal populations in rice grains. Moreover, volatiles produced by these two strains significantly reduced aflatoxin production in the rice grains by A. flavus. To our knowledge, this is the first report of the suppression of A. flavus aflatoxin production in rice grains using B. megaterium and P. protegens volatiles.

Evaluation of cnpt-direct assay and development of a modified cnpt-edta assay todifferentiatendm from oxa-48 like carbapenemases

Infections caused by carbapenemase producing bacteria are a worldwide threat, due to their association with high mortality. Early diagnosis is the key to treatment and can help in preventing the spread of infection. In this study, we have evaluated the phenotypic assay, carbaNP test (CNPt)-direct, that is used to identify carbapenemase producers. We have introducedCNPt-EDTA direct, a modification of the (CNPt)-direct test. The CNPt-EDTA direct can be used to distinguish strong metallo carbapenemases producers such as the NDM enzyme from weak hydrolyzers such as the OXA-48 like enzymes.Thirty-one bacterial isolates, including twenty eight clinical isolates, two positive control strains and one negative control strain, were subjected to the CNPt- direct, and the CNPt-EDTA-direct assays. Based on the color produced(from red to yellow or orange), twenty seven clinical isolates tested carbapenemase positive by CNPt-direct, of which eighteen were strong carbapenemase hydrolyzers and nine weak hydrolyzers. Each isolate was then tested by CNPt-EDTA direct and confirmed by gene specific PCR. When analysed by the CNPt-EDTA-direct, the strong carbapenem hydrolysers showed that fourteen isolates harboured the NDM enzyme, while four harboured a strong hydrolyser in association with or without class B enzyme. Out of the nine weak hydrolysers, eight were EDTA insensitive and harboured the OXA-48 like enzyme and one harboured the VIM enzyme. Thus the use of CNPt-direct in conjunction with CNPt-EDTA direct is a cost-effective and sensitive method to identify and classify carbapenemases, especially the NDM and OXA-48 like enzymes.

Antifungal sourdough lactic acid bacteria as biopreservation tool in quinoa and rice bread

The use of sourdough fermented with specific strains of antifungal lactic acid bacteria can reduce chemical preservatives in bakery products. The main objective of this study was to investigate the production of antifungal carboxylic acids after sourdough fermentation of quinoa and rice flour using the antifungal strains Lactobacillus reuteri R29 and Lactobacillus brevis R2Δ as bioprotective cultures and the non-antifungal L. brevis L1105 as a negative control strain. The impact of the fermentation substrate was evaluated in terms of metabolic activity, acidification pattern and quantity of antifungal carboxylic acids. These in situ produced compounds (n=20) were extracted from the sourdough using a QuEChERS method and detected by a new UHPLC-MS/MS chromatography. Furthermore, the sourdough was applied in situ using durability tests against environmental moulds to investigate the biopreservative potential to prolong the shelf life of bread. Organic acid production and TTA values were lowest in rice sourdough. The sourdough fermentation of the different flour substrates generated a complex and significantly different profile of carboxylic acids. Extracted quinoa sourdough detected the greatest number of carboxylic acids (n=11) at a much higher concentration than what was detected from rice sourdough (n=9). Comparing the lactic acid bacteria strains, L. reuteri R29 fermented sourdoughs contained generally higher concentrations of acetic and lactic acid but also the carboxylic acids. Among them, 3-phenyllactic acid and 2-hydroxyisocaproic acid were present at a significant concentration. This was correlated with the superior protein content of quinoa flour and its high protease activity. With the addition of L. reuteri R29 inoculated sourdough, the shelf life was extended by 2 days for quinoa (+100%) and rice bread (+67%) when compared to the non-acidified controls. The L. brevis R2Δ fermented sourdough bread reached a shelf life of 4 days for quinoa (+100%) and rice (+33%). However, the shelf life was similar to the chemically acidified control indicating that the preservation effect of the carboxylic acids seems to have a minor contribution effect on the antifungal activity in gluten-free breads.

A Comparative Study of the Metabolic and Skeletal Response of C57BL/6J and C57BL/6N Mice in a Diet-Induced Model of Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) represents a complex clinical scenario of altered energy metabolism and increased fracture incidence. The C57BL/6 mouse model of diet-induced obesity has been used to study the mechanisms by which altered glucose homeostasis affects bone mass and quality, but genetic variations in substrains of C57BL/6 may have confounded data interpretation. This study investigated the long-term metabolic and skeletal consequences of two commonly used C57BL/6 substrains to a high fat (HF) diet. Male C57BL/6J, C57BL/6N, and the negative control strain, C3H/HeJ, mice were fed a control or HF diet for 24 wks. C57BL/6N mice on a HF diet demonstrated an increase in plasma insulin and blood glucose as early as 4 wk, whereas these responses were delayed in the C57BL/6J mice. The C57BL/6N mice exhibited more severe hepatic steatosis and inflammation. Only the C57BL/6N mice lost significant trabecular bone in response to the high fat diet. The C3H/HeJ mice were protected from bone loss. The data show that C57BL/6J and C57BL/6N mice differ in their metabolic and skeletal response when fed a HF diet. These substrain differences should be considered when designing experiments and are likely to have implications on data interpretation and reproducibility.

First Report of Bacterial Leaf Blight on Mustard Greens (Brassica juncea) Caused by Pseudomonas cannabina pv. alisalensis in Mississippi.

In 2010, a brassica leafy greens grower in Sunflower County, MS, observed scattered outbreaks of a leaf blight on mustard greens (Brassica juncea) in a 180-ha field. A severe outbreak of leaf blight occurred on mustard greens and turnip greens (B. rapa) in the same field in 2011 with more than 80 ha affected. The affected field, established in 2010, had no prior history of being cropped to brassica leafy greens. Symptoms appeared on the 6-week-old transplants as brown to tan necrotic spots with faint chlorotic borders and associated water-soaking. Lesions varied from 4 mm to 3 cm in diameter and often coalesced to cover >90% of older leaves. Whole plants of the mustard greens cv. Florida Broadleaf were collected in 2011 from the symptomatic field. Leaves were surface-disinfested with 0.5% NaOCl for 5 min, rinsed twice in sterilized distilled water [(sd)H2O], macerated in sdH2O, then streaked onto nutrient agar (NA), pseudomonas agar F (PAF), and potato dextrose agar (PDA). Little or no bacterial growth was observed on PDA, while on NA and PAF the majority of bacterial growth appeared to be a single colony type. All strains collected (25 total, one per plant) were gram-negative and fluoresced blue-green under UV light after 48 h at 28°C on PAF. All 25 strains were identified as belonging to Pseudomonas group 1a using Lelliot's determinative assay (2). Ten of the 25 strains were tested for pathogenicity on Florida Broadleaf, and turnip greens cv. Alamo. Bacteria were grown on PAF for 48 h, and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants (three plants per cultivar) were sprayed with the appropriate bacterial suspension to runoff, placed at 100% relative humidity for 48 h, and then put in a growth chamber at 28°C with a 16-h diurnal light cycle for 14 days. Additionally, three plants each of Florida Broadleaf and Alamo were either sprayed with H2O or inoculated with Pseudomonas cannabina pv. alisalensis (Pca) pathotype strain BS91 (1). All 10 strains, as well as the Pca pathotype strain, were pathogenic on both cultivars and caused symptoms similar to those observed in the field. Symptoms were not observed on H2O-sprayed plants. Comparative rep-PCR analysis using the BOXA1R primer showed the 10 strains had identical DNA-banding profiles and were identical to that of Pca BS91 (5). Five strains tested using a Pca-specific, 'light-tagged' reporter bacteriophage gave a strong positive reaction, while a negative control strain, P. syringae pv. maculicola, gave no signal (3). From these tests, the isolated bacteria were determined to be Pca. Bacteria re-isolated on PAF from the inoculated Florida Broadleaf plants had identical rep-PCR profiles with those of the strains used for inoculations. Over the past 10 years, Pca has been found in numerous states in the United States, as well as in Europe, Australia, and Japan (4). As brassica leafy greens production expands to new fields and new states, leaf blight caused by Pca appears to become a problem rapidly. Since resistant cultivars and highly effective bactericides are lacking, growers are extremely concerned about the rapid spread of this disease into existing and new brassica leafy greens regions.

Lactic acid bacteria bioprotection applied to the malting process. Part I: Strain characterization and identification of antifungal compounds

Lactic acid bacteria (LAB) are of interest to cereal and beverage industry due to their contribution to the microbial safety and quality of fermented food/beverages. The main objectives of this study were to optimize the production of an antifungal cell-free-supernatant (cfs), based on lactic acid bacteria, using a wort-base substrate; and the subsequent identification of its active acid-base antifungal compounds. Two antifungal strains (Lactobacillus amylovorus DSM19280 and Lactobacillus reuteri R29) and a negative control strain (L. amylovorus DSM20552) were used. Barley based malt extract (wort) was produced and used as a fermentation substrate. Worts fermented with LAB were characterized in detail (total cell counts, pH, and total titratable acids (TTA) over time (0–120 h). A microtiter plate and an overlay agar based assay were used to evaluate the antifungal activity of the LAB cfs. Sugars, organic acids of the fermented and non-fermented wort (control) were analysed by HPLC. LAB antifungal metabolites were quantified using a QuEChERS method and an HPLC-UV/PDA system. Results show that wort produced from barley malt is a suitable substrate for LAB and dependent on the species and the fermentation time. The type of antifungal compounds can vary significantly. L. amylovorus DSM19280 cfs inhibited Fusarium culmorum spores at levels of 104 spores.mL−1, for 7 days, whereas L. reuteri R29 cfs inhibited up to 105 spores.mL−1 for the same time. Thirteen acid-base antifungal compounds were identified in L. reuteri R29 cfs and twelve in L. amylovorus DSM19280. Among them, phenyllactic, OH-phenylactic and benzoic acids were present at a significant concentration. This study demonstrates that the LAB wort-base cfs exhibits potent antifungal activity and is the ideal substrate for applications in a wide range of cereal products.

Biocontrol activity and root colonization by Pseudomonas corrugata strains CCR04 and CCR80 against Phytophthora blight of pepper

Previously, we selected Pseudomonas corrugata strains CCR04 and CCR80 as rhizobacteria suppressive to Phytophthora blight of pepper caused by Phytophthora capsici. In this study, we investigated soil microbial activity in pepper plants root-drenched with strains CCR04 and CCR80 in relation to their biocontrol activity, root colonization by using bacterial population counts and scanning electron microscopy, biofilm formation and cell motility as well as cell sensitivity to hydrogen peroxide (H2O2). As a result, strains CCR04 and CCR80 more effectively suppressed disease expression in pepper plants through root colonization than did Paenibacillus polymyxa AC-1 (positive control), Escherichia coli DH5α (negative control) or MgSO4 solution (untreated control). Strains CCR04 and CCR80 had efficient biofilm formation and cell motility (swimming and swarming activities) abilities and responded to certain tested compounds (amino acids, organic acids and sugars), which can be found in root exudates. Strains CCR04 and CCR80 and the positive control strain AC-1 were relatively insensitive to H2O2, a reactive oxidative species at concentration up to 20 mM, unlike the negative control strain DH5α. Taken together, these results suggest that P. corrugata CCR04 and CCR80 can effectively inhibit P. capsici infection of pepper plants through successful colonization of plant roots. This bacterial colonization may be facilitated by the biofilm formation ability and cell motility in addition to reduced sensitivity to H2O2 and probably the production of antimicrobial compounds. These findings highlight the potential of strains CCR04 and CCR80 as biocontrol agents for the management of Phytophthora blight of pepper.

Extraction of water-soluble xylan from wheat bran and utilization of enzymatically produced xylooligosaccharides by Lactobacillus, Bifidobacterium and Weissella spp.

Xylan was extracted from wheat bran after heat pretreatment in water using either an autoclave or a microwave oven. Xylooligosaccharides (XOS) were produced from the xylan using the thermostable xylanase RmXyn10A and the potential prebiotic properties of XOS were studied in vitro with different human gut bacteria: Lactobacillus brevis (DSMZ 1269), Bifidobacterium adolescentis (ATCC 15703) and two strains of recently isolated lactic acid bacteria from the species pair Weissella cibaria/confusa. The highest yield of (arabino)xylan with the heat pretreatment was obtained at 185 °C for 10 min. Higher temperature led to fewer arabinose substitutions present on the backbone which in turn resulted in a slightly more efficient enzymatic hydrolysis by RmXyn10A.Using the produced XOS hydrolysate as carbon source, xylobiose uptake was confirmed for all bacterial species studied while xylotriose uptake could be confirmed for B. adolescentis and the Weissella strains. The negative control strain Escherichia coli (BL 21) did not use XOS as a carbon source. L. brevis, B. adolescentis and the Weissella spp. all showed growth on XOS, verified by increases in cell density, lactic acid and acetic acid production after 48 h incubation. Corresponding increases were not found using the non-hydrolysed xylan as carbon source.

Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

Increasing the production of fatty acids by microbial fermentation remains an important step toward the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16°C, 3.5-fold at 22°C and 1.5-fold at 30°C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations toward accessible biodiesel precursors.

Gene expression cross-profiling in genetically modified industrial Saccharomyces cerevisiae strains during high-temperature ethanol production from xylose

Production of ethanol from xylose at high temperature would be an economical approach since it reduces risk of contamination and allows both the saccharification and fermentation steps in SSF to be running at elevated temperature. Eight recombinant xylose-utilizing Saccharomyces cerevisiae strains developed from industrial strains were constructed and subjected to high-temperature fermentation at 38°C. The best performing strain was sun049T, which produced up to 15.2g/L ethanol (63% of the theoretical production), followed by sun048T and sun588T, both with 14.1g/L ethanol produced. Via transcriptomic analysis, expression profiling of the top three best ethanol producing strains compared to a negative control strain, sun473T, led to the discovery of genes in common that were regulated in the same direction. Identification of the 20 most highly up-regulated and the 20 most highly down-regulated genes indicated that the cells regulate their central metabolism and maintain the integrity of the cell walls in response to high temperature. We also speculate that cross-protection in the cells occurs, allowing them to maintain ethanol production at higher concentration under heat stress than the negative controls. This report provides further transcriptomics information in the interest of producing a robust microorganism for high-temperature ethanol production utilizing xylose.

A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes

The development of renewable alternatives to diesel and jet fuels is highly desirable for the heavy transportation sector, and would offer benefits over the production and use of short-chain alcohols for personal transportation. Here, we report the development of a metabolically engineered strain of Escherichia coli that overproduces medium-chain length fatty acids via three basic modifications: elimination of beta-oxidation, overexpression of the four subunits of acetyl-CoA carboxylase, and expression of a plant acyl-acyl carrier protein (ACP) thioesterase from Umbellularia californica (BTE). The expression level of BTE was optimized by comparing fatty acid production from strains harboring BTE on plasmids with four different copy numbers. Expression of BTE from low copy number plasmids resulted in the highest fatty acid production. Up to a seven-fold increase in total fatty acid production was observed in engineered strains over a negative control strain (lacking beta-oxidation), with a composition dominated by C(12) and C(14) saturated and unsaturated fatty acids. Next, a strategy for producing undecane via a combination of biotechnology and heterogeneous catalysis is demonstrated. Fatty acids were extracted from a culture of an overproducing strain into an alkane phase and fed to a Pd/C plug flow reactor, where the extracted fatty acids were decarboxylated into saturated alkanes. The result is an enriched alkane stream that can be recycled for continuous extractions. Complete conversion of C(12) fatty acids extracted from culture to alkanes has been demonstrated yielding a concentration of 0.44 g L(-1) (culture volume) undecane.

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis.

A new protein (SA-5K) secreted in culture filtrates by Mycobacterium bovis, Mycobacterium tuberculosis, and few other mycobacterial species was previously identified and purified in our laboratory. In order to evaluate the putative role of SA-5K during intracellular mycobacterial growth, in the present study the SA-5K gene was cloned and expressed in Mycobacterium smegmatis, a rapid growing non-pathogenic mycobacterium which does not contain the gene for the protein. SA-5K expression in the THP-1 human macrophage cell line infected with the recombinant strain (M. smegmatis-pROL5K) was demonstrated by RT-PCR on RNA extracted from bacterial cells following 24 and 48 h of infection. Intracellular SA5K expression was associated with a higher cfu increase of M. smegmatis-pROL5K in comparison to the negative control strain (M. smegmatis recombinant for the cloning vector) (P=0.01). No significant change in SA-5K synthesis by M. smegmatis-pROL5K was observed when the recombinant strain was grown in vitro in different stress conditions such as iron deprivation, pH 4.5, presence of nitric oxide or hydrogen peroxide. The results presented in this study suggest a possible role for SA-5K in intracellular survival of recombinant M. smegmatis, though the function of the protein remains unknown.