Characterization of Phenotypic Changes inPseudomonas putida in Response to Surface-Associated Growth

ABSTRACT The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment ofP. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P.putida and pure C12-HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate thatP. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.

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Comparative Proteomics Reveals the Spoilage-Related Factors of Shewanella putrefaciens Under Refrigerated Condition

Frontiers in Microbiology ◽

10.3389/fmicb.2021.740482 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhengkai Yi ◽

Jing Xie

Keyword(s):

Amino Acid ◽

Proteomic Analysis ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Sulfur Metabolism ◽

Shewanella Putrefaciens ◽

Extracellular Proteins ◽

Differentially Expressed ◽

Differentially Expressed Protein ◽

Fish Proteins

Shewanella putrefaciens is a microorganism with strong spoilage potential for aquatic products. This study aimed to investigate the potential spoilage factors of S. putrefaciens by comparative proteomic analysis. The spoilage potential of two strains of S. putrefaciens (00A and 00B) isolated from chilled spoiled bigeye tuna was investigated. The results of total volatile basic nitrogen (TVB-N), trimethylamine (TMA) in fish inoculated with S. putrefaciens, extracellular protease activity of S. putrefaciens, and degradation of fish proteins indicated that the spoilage potential of S. putrefaciens 00A was much higher than that of 00B. Fish proteins are usually degraded by spoilage microorganism proteases into small molecular peptides and amino acids, which are subsequently degraded into spoilage metabolites in bacterial cells, leading to deterioration of fish quality. Thus, proteomic analysis of the extracellular and intracellular proteins of 00A vs. 00B was performed. The results indicated that the intracellular differentially expressed protein (IDEP) contained 243 upregulated proteins and 308 downregulated proteins, while 78 upregulated proteins and 4 downregulated proteins were found in the extracellular differentially expressed protein (EDEP). GO annotation revealed that IDEP and EDEP were mainly involved in cellular and metabolic processes. KEGG annotation results showed that the upregulated proteins in IDEP were mainly involved in sulfur metabolism, amino acid metabolism, and aminoacyl-tRNA biosynthesis, while downregulated proteins were related to propanoate metabolism. In contrast, EDEP of KEGG annotation was mainly involved in ribosomes, quorum sensing, and carbohydrate metabolism. Proteins associated with spoilage containing sulfur metabolism (sulfite reductase, sulfate adenylyltransferase, adenylyl-sulfate kinase), amino acid metabolism (biosynthetic arginine decarboxylase, histidine ammonia-lyase), trimethylamine metabolism (trimethylamine-N-oxide reductase), and extracellular proteins (ATP-dependent Clp protease proteolytic subunit) were identified as upregulated. These proteins may play a key role in the spoilage potential of S. putrefaciens. These findings would contribute to the identification of key spoilage factors and understanding of the spoilage mechanism of microorganisms.

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Comparative Proteomics Reveals Cold Acclimation Machinery Through Enhanced Carbohydrate and Amino Acid Metabolism in Wucai (Brassica Campestris L.)

Plants ◽

10.3390/plants8110474 ◽

2019 ◽

Vol 8 (11) ◽

pp. 474

Author(s):

Lingyun Yuan ◽

Shilei Xie ◽

Libing Nie ◽

Yushan Zheng ◽

Jie Wang ◽

...

Keyword(s):

Amino Acid ◽

Cold Acclimation ◽

Low Temperatures ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Expression Patterns ◽

Differentially Expressed ◽

Pcr Analysis ◽

Differentially Expressed Proteins ◽

Limited Information

Limited information is available on the cold acclimation of non-heading Chinese cabbage (NHCC) under low temperatures. In this study, the isobaric tags for relative and absolute quantification (iTRAQ) were used to illustrate the molecular machinery of cold acclimation. Compared to the control (Cont), altogether, 89 differentially expressed proteins (DEPs) were identified in wucai leaves responding to low temperatures (LT). Among these proteins, 35 proteins were up-regulated ((and 54 were down-regulated). These differentially expressed proteins were categorized as having roles in carbohydrate metabolism, photosynthesis and energy metabolism, oxidative defense, amino acid metabolism, metabolic progress, cold regulation, methylation progress, and signal transduction. The fructose, glucose, and sucrose were dramatically increased in response to cold acclimation. It was firstly reported that aspartate, serine, glutamate, proline, and threonine were significantly accumulated under low temperatures. Results of quantitative real-time PCR analysis of nine DEPs displayed that the transcriptional expression patterns of six genes were consistent with their protein expression abundance. Our results demonstrated that wucai acclimated to low temperatures through regulating the expression of several crucial proteins. Additionally, carbohydrate and amino acid conversion played indispensable and vital roles in improving cold assimilation in wucai.

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POS0421 COMBINED ANALYSIS OF METABOLIC AND TRANSCRIPTOMIC KIDNEY PROFILES OF NZW/B-F1 MURINE LUPUS UNCOVERS BIOLOGICAL MECHANISMS PRECEDING THE ONSET OF NEPHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.4115 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 439.2-440

Author(s):

T. Manolakou ◽

I. Tsiara ◽

D. Nikolopoulos ◽

P. Garantziotis ◽

D. Benaki ◽

...

Keyword(s):

Amino Acid ◽

Differentially Expressed Genes ◽

Metabolic Pathways ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Univariate Analysis ◽

Differentially Expressed ◽

Disease Stage ◽

Cellular Respiration ◽

H Nmr

Background:Metabolic pathways are important regulators of immune differentiation and activation in kidneys. Kidneys directly impact systemic metabolism, circulating metabolite levels, and express intrinsic metabolic activity. The integration of renal metabolomic and transcriptomic profiles may unravel unique gene-metabolite pairs of biological significance in lupus nephritis (LN).Objectives:To decipher gene-metabolite signatures at both pre-nephritic and nephritic stages of lupus.Methods:Kidneys were isolated and snap-frozen after perfusion from female NZB/NZW-F1 lupus mice at the pre-nephritic (3-month-old) and nephritic (6-month-old exhibiting ≥100 ng/dL of urine protein) stage of lupus (n=6/group). Age-matched female C57BL/6 mice were used as healthy controls. Sample extracts were used for RNA sequencing and 1H-NMR spectroscopy metabolic profiling. DESeq2 was used to identify differentially expressed genes. Univariate analysis was used to reveal metabolic differences characteristic for nephritis.Results:Comparative transcriptomic analyses uncovered multiple transcripts related to metabolic pathways: In pre-nephritic kidneys, lipid metabolism, cellular respiration, TCA cycle, amino acid metabolism processes were overrepresented in the upregulated genes while in nephritic kidneys, amino acid metabolism processes were overrepresented among the downregulated genes (Figure 1). 1H-NMR analysis revealed a total of 49 metabolites. Comparison of the metabolic levels of nephritic and pre-nephritic animals revealed that ADP, ATP, NAD+, Taurine and Myo-inositol decreased, while Thr increased significantly. The comparison to corresponding control animals, demonstrated that only myo-inositol increased significantly. Integration of kidney metabolomics and transcriptomics indicated the involvement of processes related to glutathione metabolism, leukocyte trans-endothelial migration and antigen presentation during the established renal disease stage.Conclusion:The combined transcriptomics and metabolomics analysis revealed metabolic derangements in lupus-affected kidneys both during subclinical and overt LN. Deregulated tissue-levels of taurine and myo-inositol at the subclinical stage of the disease suggest aberrant renal biochemistry preceding the development of overt LN that may directly impact systemic metabolism and circulating metabolite levels.Figure 1.Pathways linked to cell metabolism were overrepresented among 3-month upregulated and 6-month lupus mice (F1) downregulated DEGS (differentially expressed genes) compared to controls (C57BL/6).Acknowledgements:This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 742390).Disclosure of Interests:None declared

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Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress

PLoS ONE ◽

10.1371/journal.pone.0245494 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0245494

Author(s):

Yan Fang ◽

Jeffrey A. Coulter ◽

Junyan Wu ◽

Lijun Liu ◽

Xuecai Li ◽

...

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Cold Stress ◽

Cold Tolerance ◽

Brassica Rapa ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Differentially Expressed ◽

Serine Acetyltransferase ◽

Turnip Rape

Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two ω-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), δ-ornithine aminotransferase (δ-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.

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557. Impact of Heavy Metal Exposure in the Transcriptional Response of Methicillin--Resistant Staphylococcus aureus (MRSA)-USA300 Latin-American Variant (USA300-LV)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.626 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S264-S265

Author(s):

Aura M Echeverri ◽

Sandra Rincon ◽

Sebastian Solano ◽

Rafael Rios ◽

Lina P Carvajal ◽

...

Keyword(s):

Immune System ◽

Amino Acid ◽

Functional Groups ◽

Differentially Expressed Genes ◽

Unknown Function ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Differentially Expressed ◽

Host Immune System ◽

Qrt Pcr

Abstract Background USA 300-LV is the predominant MRSA clone in Colombia and contains a genomic island designated “COMER” with genes for copper (Cu) and mercury (Hg) resistance. HM environmental contamination is a serious threat to public health in Colombia and could also influence the selection and evolution of HM resistance genes in MRSA. Here, we investigate the global transcriptomic responses of USA300-LV after exposure to HM under the hypothesis that USA300-LV strains are highly capable of sustaining higher HM concentrations Methods We performed comparative RNAseq experiments in USA300-LV clinical strain (CA-MRSA12). Total RNA was isolated in exponential phase in the absence and presence of sub-inhibitory concentrations of Cu and Hg (3 replicates). cDNA libraries were prepared and sequenced on an Illumina platform. Differentially expressed genes (DEG) were calculated by DeSeq2 (p-adjusted value ˂ 0.01) and results on 19 selected genes were confirmed by qRT-PCR. Results US300-LV exhibited a larger number of differentially expressed genes when exposed to Hg (n = 114) compared with Cu treatment (n = 16). The most common functional groups of genes upregulated after Hg exposure included those involved in amino acid metabolism (n = 18). In contrast, 45 genes were downregulated after Hg exposure, mostly associated to host immune system defense (n = 11). qRT-PCR confirmed that the most upregulated genes were those involved in murein hydrolase activity, Hg resistance and the transcriptional regulator Cro/CI. Of 9 genes that were downregulated, functional groups included ype VII secretion system, immune modulators and leucocidins. Copper treatment resulted in only 12 genes that were upregulated including those in the COMER element (n = 6), aminoacid metabolism (n = 3), ROS response (n = 1), host immune system defense (n = 1) and unknown function (n = 1). Downregulated genes were those associated to host immune system defense (n = 2), energy generation (n = 1) and unknown function (n = 1). Conclusion Differential adaptive responses after exposure to HM in USA300-LV suggest a role in the evolution of antimicrobial resistance and successful spread in the region. Metabolic adaptations involving amino acid metabolism seem to play a role in the evolution of HM resistance in MRSA. Disclosures All authors: No reported disclosures.

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