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Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently (or robustly) with respect to cellular growth phase (exponential and stationary) and fermentation scale (microtiter plates, tubes, flasks, and bioreactors). However, nearly all endogenous promoters (especially in Saccharomyces cerevisiae) do not perform in this manner. In this work, a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions. Using a multi-dimensional RNA-seq dataset, promoters with specific phase dependencies were identified. Fragments enriched with functional transcription factors were identified using MEME suite. These motif-containing fragments could impart activity dependence in the opposing condition. Specifically, we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively. Further, we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks. Overall, this work presents and validates a new strategy for engineering promoters in S. cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture. [ABSTRACT FROM AUTHOR]

Objective: In the realm of Class I pathogens, Burkholderia pseudomallei (BP) stands out for its propensity to induce severe pathogenicity. Investigating the intricate interactions between BP and host cells is imperative for comprehending the dynamics of BP infection and discerning biomarkers indicative of the host cell response process. Methods: mRNA extraction from BP-infected mouse macrophages constituted the initial step of our study. Employing gene expression arrays, the extracted RNA underwent conversion into digital signals. The percentile shift method facilitated data processing, with the identification of genes manifesting significant differences accomplished through the application of the t-test. Subsequently, a comprehensive analysis involving Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway was conducted on the differentially expressed genes (DEGs). Leveraging the ESTIMATE algorithm, gene signatures were utilized to compute risk scores for gene expression data. Support vector machine analysis and gene enrichment scores were instrumental in establishing correlations between biomarkers and macrophages, followed by an evaluation of the predictive power of the identified biomarkers. Results: The functional and pathway associations of the DEGs predominantly centered around G protein-coupled receptors. A noteworthy positive correlation emerged between the blue module, consisting of 416 genes, and the StromaScore. FZD4, identified through support vector machine analysis among intersecting genes, indicated a robust interaction with macrophages, suggesting its potential as a robust biomarker. FZD4 exhibited commendable predictive efficacy, with BP infection inducing its expression in both macrophages and mouse lung tissue. Western blotting in macrophages confirmed a significant upregulation of FZD4 expression from 0.5 to 24 h post-infection. In mouse lung tissue, FZD4 manifested higher expression in the cytoplasm of pulmonary epithelial cells in BP-infected lungs than in the control group. Conclusion: These findings underscore the upregulation of FZD4 expression by BP in both macrophages and lung tissue, pointing to its prospective role as a biomarker in the pathogenesis of BP infection. [ABSTRACT FROM AUTHOR]

Background: Serratia ureilytica DW2 is a highly efficient phosphate-solubilizing bacteria isolated from Codonopsis pilosula rhizosphere soil that can promote the growth of C. pilosula; nonetheless, until now, no validated reference genes from the genus Serratia have been reported that can be used for the normalization of quantitative real-time polymerase chain reaction (RT--qPCR) data. Methods: To screen stable reference genes of S. ureilytica DW2, the expression of its eight candidate reference genes (16S rRNA, ftsZ, ftsA, mreB, recA, slyD, thiC, and zipA) under different treatment conditions (pH, temperature, culture time, and salt content) was assayed by RT--qPCR. The expression stability of these genes was analyzed using different algorithms (geNorm, NormFinder, and BestKeeper). To verify the reliability of the data, the expression of the glucose dehydrogenase (gdh) gene under different soluble phosphate levels was quantified using the most stably expressed reference gene. Results: The results showed that the zipA and 16S rRNA genes were the most stable reference genes, and the least stable genes were thiC and recA. The expression of gdh was consistent with the phosphate solubilization ability on plates containing the National Botanical Research Institute phosphate growth medium. Conclusion: Therefore, this study provides a stable and reliable reference gene of Serratia for the accurate quantification of functional gene expression in future studies. [ABSTRACT FROM AUTHOR]

Iron is a limiting factor in such a condition that usually is sequestered by the host during polymicrobial infections of Pseudomonas aeruginosa and Staphylococcus aureus. This study aimed to investigate the interaction of S. aureus and P. aeruginosa, which alters iron-related sigma factors regulation and antibiotic resistance. The antibiotic resistance of P. aeruginosa and S. aureus was investigated in a L929 cell culture model. The expression level of pvdS, hasI (P. aeruginosa sigma factors), and sigS (S. aureus sigma factor) genes was determined using Quantitative Real-Time PCR. pvdS and hasI were downregulated during co-culture with S. aureus, while the susceptibility to carbapenems increased (p-value < 0.0001). Also, there was a direct significant relationship between resistance to vancomycin with sigS. Regarding the findings of the current study, iron-related sigma factors of P. aeruginosa and S. aureus play a role in induction susceptibility to various antibiotics, including carbapenems and vancomycin. [ABSTRACT FROM AUTHOR]

Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety. [ABSTRACT FROM AUTHOR]

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC's. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen. [ABSTRACT FROM AUTHOR]

Because of the ability to metabolize a large number of electron acceptors such as nitrate, nitrite, fumarate, and metal oxides, Shewanella species have attracted much attention in recent years. Generally, the use of these electron acceptors is mainly achieved through electron transfer proteins and their interactions which will dynamically change across different environmental conditions in cells. Therefore, functional analysis of condition-specific molecular networks can reveal biological information on electron transfer processes. By integrating expression data and molecular networks, we constructed condition-specific molecular networks for Shewanella piezotolerans WP3. We then identified condition-specific key genes and studied their potential functions with an emphasis on their roles in electron transfer processes. Functional module analysis showed that different flagellar assembly modules appeared under these conditions and suggested that flagellar proteins are important for these conditions. We also identified the electron transfer modules underlying these various environmental conditions. The present results could help with screening electron transfer genes and understanding electron transfer processes under various environmental conditions for the Shewanella species. [ABSTRACT FROM AUTHOR]

Detailed whole-cell modeling requires an integration of heterogeneous cell processes having different modeling formalisms, for which whole-cell simulation could remain tractable. Here, we introduce BiPSim, an open-source stochastic simulator of template-based polymerization processes, such as replication, transcription and translation. BiPSim combines an efficient abstract representation of reactions and a constant-time implementation of the Gillespie's Stochastic Simulation Algorithm (SSA) with respect to reactions, which makes it highly efficient to simulate large-scale polymerization processes stochastically. Moreover, multi-level descriptions of polymerization processes can be handled simultaneously, allowing the user to tune a trade-off between simulation speed and model granularity. We evaluated the performance of BiPSim by simulating genome-wide gene expression in bacteria for multiple levels of granularity. Finally, since no cell-type specific information is hard-coded in the simulator, models can easily be adapted to other organismal species. We expect that BiPSim should open new perspectives for the genome-wide simulation of stochastic phenomena in biology. [ABSTRACT FROM AUTHOR]

Increased tolerance to light stress in cyanobacteria is a desirable feature for their applications. Here, we obtained a high light tolerant (Tol) strain of Synechocystis sp. PCC6803 through an adaptive laboratory evolution, in which the cells were repeatedly sub-cultured for 52 days under high light stress conditions (7000 to 9000 μmol m−2 s−1). Although the growth of the parental strain almost stopped when exposed to 9000 μmol m−2 s−1, no growth inhibition was observed in the Tol strain. Excitation-energy flow was affected because of photosystem II damage in the parental strain under high light conditions, whereas the damage was alleviated and normal energy flow was maintained in the Tol strain. The transcriptome data indicated an increase in isiA expression in the Tol strain under high light conditions. Whole genome sequence analysis and reverse engineering revealed two mutations in hik26 and slr1916 involved in high light stress tolerance in the Tol strain. In this paper, the authors investigated the high light stress response in Synechocystis sp. PCC6803 and revealed two key mutations in hik26 and slr1916 using whole genome sequencing and reverse engineering. Here, the high light tolerant strain exhibited no growth inhibition in conditions up to 9000 μmol m−2 s−1, which had no effect on excitation-energy flow and instead showed an increase in isiA expression. [ABSTRACT FROM AUTHOR]

Growth of plants in soil inoculated with plant growth promoting bacteria (PGPB) producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase or expression of the corresponding acdS gene in transgenic lines reduces the decline in shoot length, shoot weight and photosynthetic capacity triggered by salt stress in Camelina sativa. Reducing the levels of ethylene attenuated the salt stress response as inferred from decreases in the expression of genes involved in development, senescence, chlorosis and leaf abscission that are highly induced by salt to levels that may otherwise have a negative effect on plant growth and productivity. Growing plants in soil treated with Pseudomonas migulae 8R6 negatively affected ethylene signaling, auxin and JA biosynthesis and signalling, but had a positive effect on the regulation of genes involved in GA signaling. In plants expressing acdS, the expression of the genes involved in auxin signalling was positively affected, while the expression of genes involved in cytokinin degradation and ethylene biosynthesis were negatively affected. Moreover, fine-tuning of ABA signaling appears to result from the application of ACC deaminase in response to salt treatment. Moderate expression of acdS under the control of the root specific rolD promoter or growing plants in soil treated with P. migulae 8R6 were more effective in reducing the expression of the genes involved in ethylene production and/or signaling than expression of acdS under the more active Cauliflower Mosaic Virus 35S promoter. [ABSTRACT FROM AUTHOR]

Neutrophilic Fe(II) oxidizing bacteria like Mariprofundus ferrooxydans are obligate chemolithoautotrophic bacteria that play an important role in the biogeochemical cycling of iron and other elements in multiple environments. These bacteria generally exhibit a singular metabolic mode of growth which prohibits comparative "omics" studies. Furthermore, these bacteria are considered non-amenable to classical genetic methods due to low cell densities, the inability to form colonies on solid medium, and production of copious amounts of insoluble iron oxyhydroxides as their metabolic byproduct. Consequently, the molecular and biochemical understanding of these bacteria remains speculative despite the availability of substantial genomic information. Here we develop the first genetic system in neutrophilic Fe(II) oxidizing bacterium and use it to engineer lithoheterotrophy in M. ferrooxydans, a metabolism that has been speculated but not experimentally validated. This synthetic biology approach could be extended to gain physiological understanding and domesticate other bacteria that grow using a single metabolic mode. [ABSTRACT FROM AUTHOR]

The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription–translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all- E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) are among the most important biofilm-forming pathogens responsible for hard-to-treat infections. Looking for alternatives to antibiotics that prevent biofilm formation, we investigated the effects of manuka honey on the transcriptional profile of genes essential for staphylococcal biofilm formation using qRT-PCR. mRNA from two hospital MRSA strains (strong and weak biofilm producer) were isolated after 4, 8, 12 and 24 h from cells grown in biofilm. Manuka honey at 1/2 minimum biofilm inhibition concentration (MBIC) significantly reduced MRSA cell viability in biofilm. Manuka honey downregulated the genes encoding laminin- (eno), elastin- (ebps) and fibrinogen binding protein (fib), and icaA and icaD involved in biosynthesis of polysaccharide intercellular adhesin in both weakly and strongly adhering strain compared to the control (untreated biofilm). Expression levels of cna (collagen binding protein) and map/eap (extracellular adherence protein—Eap) were reduced in weakly adhering strain. The lowest expression of investigated genes was observed after 12 h of manuka honey treatment at 1/2 MBIC. This study showed that the previously unknown mechanism of manuka honey action involved inhibition of S. aureus adhesion due to reduction in expression of crucial genes associated with staphylococcal biofilm. [ABSTRACT FROM AUTHOR]

Gene expression in bacteria is a remarkably controlled and intricate process impacted by many factors. One such factor is the genomic position of a gene within a bacterial genome. Genes located near the origin of replication generally have a higher expression level, increased dosage, and are often more conserved than genes located farther from the origin of replication. The majority of the studies involved with these findings have only noted this phenomenon in a single gene or cluster of genes that was re-located to pre-determined positions within a bacterial genome. In this work, we look at the overall expression levels from eleven bacterial data sets from Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti. We have confirmed that gene expression tends to decrease when moving away from the origin of replication in majority of the replicons analysed in this study. This study sheds light on the impact of genomic location on molecular trends such as gene expression and highlights the importance of accounting for spatial trends in bacterial molecular analysis. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a gram-negative bacterium that persistently colonizes the human stomach by inducing immunoregulatory responses. We have used a novel platform that integrates a bone marrow-derived macrophage and live H. pylori co-culture with global time-course transcriptomics analysis to identify new regulatory genes based on expression patterns resembling those of genes with known regulatory function. We have used filtering criteria based on cellular location and novelty parameters to select 5 top lead candidate targets. Of these, Plexin domain containing 2 (Plxdc2) was selected as the top lead immunoregulatory target. Loss of function studies with in vivo models of H. pylori infection as well as a chemically-induced model of colitis, confirmed its predicted regulatory function and significant impact on modulation of the host immune response. Our integrated bioinformatics analyses and experimental validation platform has enabled the discovery of new immunoregulatory genes. This pipeline can be used for the identification of genes with therapeutic applications for treating infectious, inflammatory, and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Background: The European Community has adopted very restrictive policies regarding the dissemination and use of genetically modified organisms (GMOs). In fact, a maximum threshold of 0.9% of contaminating GMOs is tolerated for a "GMO-free" label. In recent years, imports of undescribed GMOs have been detected. Their sequences are not described and therefore not detectable by conventional approaches, such as PCR. Results: We developed DUGMO, a bioinformatics pipeline for the detection of genetically modified (GM) bacteria, including unknown GM bacteria, based on Illumina paired-end sequencing data. The method is currently focused on the detection of GM bacteria with – possibly partial – transgenes in pure bacterial samples. In the preliminary steps, coding sequences (CDSs) are aligned through two successive BLASTN against the host pangenome with relevant tuned parameters to discriminate CDSs belonging to the wild type genome (wgCDS) from potential GM coding sequences (pgmCDSs). Then, Bray-Curtis distances are calculated between the wgCDS and each pgmCDS, based on the difference of genomic vocabulary. Finally, two machine learning methods, namely the Random Forest and Generalized Linear Model, are carried out to target true GM CDS(s), based on six variables including Bray-Curtis distances and GC content. Tests carried out on a GM Bacillus subtilis showed 25 positive CDSs corresponding to the chloramphenicol resistance gene and CDSs of the inserted plasmids. On a wild type B. subtilis, no false positive sequences were detected. Conclusion: DUGMO detects exogenous CDS, truncated, fused or highly mutated wild CDSs in high-throughput sequencing data, and was shown to be efficient at detecting GM sequences, but it might also be employed for the identification of recent horizontal gene transfers. [ABSTRACT FROM AUTHOR]

Motivation An important task in comparative genomics is to detect functional units by analyzing gene-context patterns. Colinear syntenic blocks (CSBs) are groups of genes that are consistently encoded in the same neighborhood and in the same order across a wide range of taxa. Such CSBs are likely essential for the regulation of gene expression in prokaryotes. Recent results indicate that colinearity can be conserved across multiple operons, thus motivating the discovery of multi-operon CSBs. This computational task raises scalability challenges in large datasets. Results We propose an efficient algorithm for the discovery of cross-strand multi-operon CSBs in large genomic datasets. The proposed algorithm uses match-point arithmetic, which is scalable for large datasets of microbial genomes in terms of running time and space requirements. The algorithm is implemented and incorporated into a tool with a graphical user interface, called CSBFinder-S. We applied CSBFinder-S to data mine 1485 prokaryotic genomes and analyzed the identified cross-strand CSBs. Our results indicate that most of the syntenic blocks are exclusively colinear. Additional results indicate that transcriptional regulation by overlapping transcriptional genes is abundant in bacteria. We demonstrate the utility of CSBFinder-S to identify common function of the gene-pair PulEF in multiple contexts, including Type 2 Secretion System, Type 4 Pilus System and DNA uptake machinery. Availability and implementation CSBFinder-S software and code are publicly available at https://github.com/dinasv/CSBFinder. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Background: The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure. Results: The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing. Conclusion: The ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications. [ABSTRACT FROM AUTHOR]

• Wealth of genome data are making pan-genome analysis increasingly possible. • Pan-genome analysis is an effective approach to understand pathogenic bacteria. • Pan-genome analysis can be conducted using various computational approaches. • Pan-genome analysis generates biological insights on a microbial clade. • Pan-genome analysis helps developing prevention and treatment strategies. Biological knowledge accumulated over the decades and advances in computational methods have facilitated the implementation of pan-genome analysis that aims at better understanding of genotype-phenotype associations of a specific group of organisms. Pan-genome analysis has been shown to be an effective approach to better understand a clade of pathogenic bacteria because it helps developing various and tailored therapeutic strategies on the basis of their biological similarities and differences. Here, we review recent progress in the pan-genome analysis of pathogenic bacteria. In particular, we focus on computational tools that allow streamlined pan-genome analysis. Also, various applications of pan-genome analysis including those relevant to devising strategies for the prevention and treatment of pathogenic bacteria are reviewed. [ABSTRACT FROM AUTHOR]

Emcibacter congregatus ZYLT was isolated from a sediment sample cultured in situ in a coast located in the East China Sea. The genome of E. congregatus ZYLT was sequenced and assembled into one single circular chromosome with the size of 4,189,011 bp and G+C content of 52.6%. Genomic annotation showed that E. congregatus ZYLT had an intact Type II-C CRISPR-Cas system consists of three cas genes (cas 9, cas 1, and cas 2), 34 direct repeat sequences with the length of 36 bp, and 33 spacers. The predicted Cas 9 protein was smaller than most of existing genome editing tools. This structure might have potential in developing new gene editing system and uncovering the regulatory mechanisms of CRISPR-Cas system. Besides, the comparison between E. congregatus ZYLT and its relative species living in neritic environments unraveled some common traits of the defective strategies of these bacteria to face inshore challenges including the motility, multidrug resistance, and universal efflux pumps. [ABSTRACT FROM AUTHOR]

• Integrated bioinformatics system for identifying operons in bacterial genomes. • Combining computational and experimental data improves accuracy of operon prediction. • Provides insight into co-expressed genes, functional relationships, and regulatory networks. An operon is a set of neighboring genes in a genome that is transcribed as a single polycistronic message. Genes that are part of the same operon often have related functional roles or participate in the same metabolic pathways. The majority of all bacterial genes are co-transcribed with one or more other genes as part of a multi-gene operon. Thus, accurate identification of operons is important in understanding co-regulation of genes and their functional relationships. Here, we present a computational system that uses RNA-seq data to determine operons throughout a genome. The system takes the name of a genome and one or more files of RNA-seq data as input. Our method combines primary genomic sequence information with expression data from the RNA-seq files in a unified probabilistic model in order to identify operons. We assess our method's ability to accurately identify operons in a range of species through comparison to external databases of operons, both experimentally confirmed and computationally predicted, and through focused experiments that confirm new operons identified by our method. Our system is freely available at https://cs.wellesley.edu/~btjaden/Rockhopper/. [ABSTRACT FROM AUTHOR]

In the last decade, the implementation of high‐throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon. [ABSTRACT FROM AUTHOR]

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides. [ABSTRACT FROM AUTHOR]

Acidithiobacillus ferrooxidans (At. f) is a bacterium involved in the bioleaching process. In the present study, we investigated the effects of Acidithiobacillus sp. FJ2 induced with diethyl sulfate (DES) as mutagen on the bioleaching of low grade uranium ore. The comparison was carried out within 5, 10, 15, 25 and 50% uranium ore pulp densities in the bioleaching system. The impact of the induction of Acidithiobacillus sp. FJ2 was determined by Eh and pH values, concentrations of Fe2+ and uranium extraction rates at 24 h intervals. The results showed that DES with 0.8% may lead to an obvious change on bacteria leading to improvement of bioleaching capability in 5, 10, 15% pulp densities. However, the bioleaching activity of the original bacteria was more efficient than DES-induced bacteria at 25 and 50% pulp densities. The gene expression results in 0.8% DES treated bacteria indicated that the bacteria attempt to adjust in the bioleaching systems (with different pulp densities) through decreased cyc2 and increased rus, cyc1 and coxB levels. These results suggest that uranium may induce oxidative stress in the wild and treated strains in the high pulp density, while the bacteria tried to survive and gain more energy from the iron oxidation. However, when the amount of uranium increased, the mutants couldn’t cope up woth the enhanced stress in 25 and 50% pulp densities. It may be due to inhibitory effect of uranium toxicity on adaptive processes which may change the trends. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria play a vital role as starter cultures in the fermentation of a variety of foods, often altering the flavor and texture in addition to aiding in preservation. Their importance to the industry has made them targets for metabolic engineering to improve relevant phenotypes and several methods of genome engineering in these organisms have been established. While the efficiency of these techniques is variable, in recent years the ability to select for successful recombinants has markedly increased the throughput of genome engineering. This review summarizes recent advances in genome engineering in lactic acid bacteria and applications that will be enabled by high-throughput techniques. [ABSTRACT FROM AUTHOR]

The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H2O2 and removal of the H2O2-sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H2O2. Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H2O2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons. [ABSTRACT FROM AUTHOR]

Extra Cytoplasmic Function (ECF) σ factor/regulatory protein (anti-σ factor) pairs govern environment mediated changes in gene expression in bacteria. The release of the ECF σ factor from an inactive σ/anti-σ factor complex is triggered by specific environmental stimuli. The free σ factor then associates with the RNA polymerase and drives the expression of genes in its target regulon. Multiple ECF σ/anti-σ pairs ensure calibrated changes in the expression profile by correlating diverse environmental stimuli with changes in the intracellular levels of different ECF σ factors. Specificity in σ/anti-σ factor interaction is thus essential for accurate signal transduction. Here we describe experiments to evaluate interactions between different M. tuberculosis σ and anti-σ proteins in vitro. The interaction parameters suggest that cross-talk between non-cognate σ/anti-σ pairs is likely. The sequence and conformational determinants that govern interaction specificity in a σ/anti-σ complex are not immediately evident due to substantial structural conservation. Sequence-structure analysis of all σ/anti-σ pairs suggest that conserved residues are not the primary determinants of σ/anti-σ interactions-a finding that suggests a potential route to set tolerance limits in interaction specificity. Non-specific σ/anti-σ interactions are likely to be biologically significant as it can contribute to heterogeneity in cellular responses in a bacterial population under less stringent requirements. This finding is relevant for synthetic biology approaches to engineer bacteria using σ/anti-σ transcription initiation modules for diverse applications in biotechnology. • Non-specific interactions with comparable affinity to cognate interactions are feasible in σ/anti-σ factor proteins. • Conserved residues are not the primary determinants of σ/anti-σ factor interactions. • Non-specific σ/anti-σ factor interactions are likely to contribute to heterogeneity in a bacterial population. [ABSTRACT FROM AUTHOR]

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens. Author summary: Bacterial pathogens closely survey the ambient conditions and induce virulence genes only at appropriate conditions. Upon host contact, many pathogens secrete toxins in order to subvert host defense systems. We find that such a secreted toxin in enteropathogenic Yersinia pseudotuberculosis is produced only at host body temperature. This regulation depends on a temperature-responsive RNA structure, an RNA thermometer, in the 5'-untranslated region of the toxin mRNA, which prevents translation at low temperatures when the bacterium is outside the host. Preventing melting of the RNA structure at 37°C by nucleotide substitutions that stabilize base pairing resulted in avirulent Yersinia strains unable to infect mice. Given that similar RNA thermometer-like structures exist upstream of related toxin genes in various bacterial pathogens, we propose that RNA thermometer-mediated toxin production is an evolutionary conserved mechanism. Interfering with opening of such regulatory structures might thus be a promising strategy targeting a broad spectrum of bacterial pathogens. [ABSTRACT FROM AUTHOR]

Extreme factors such as space microgravity, radiation, and magnetic field differ from those that occur on Earth. Microgravity may induce and select some microorganisms for physiological, metabolic, and/or genetic variations. This study was conducted to determine the effects of simulated microgravity conditions on the metabolism and gene expression of the probiotic bacterium Lactobacillus reuteri DSM17938. To investigate microbial response to simulated microgravity, two devices—the rotating wall vessel (RWV) and the random positioning machine (RPM)—were used. Microbial growth, reuterin production, and resistance to gastrointestinal passage were assessed, and morphological characteristics were analyzed by scanning electron microscopy. The expression of some selected genes that are responsive to stress conditions and to bile salts stress was evaluated through real-time quantitative polymerase chain reaction assay. Monitoring of bacterial growth, cell size, and shape under simulated microgravity did not reveal differences compared with 1 × g controls. On the contrary, an enhanced production of reuterin and a greater tolerance to the gastrointestinal passage were observed. Moreover, some stress genes were upregulated under RWV conditions, especially after 24 h of treatment, whereas RPM conditions seemed to determine a downregulation over time of the same stress genes. These results show that simulated microgravity could alter some physiological characteristics of L. reuteri DSM17938 with regard to tolerance toward stress conditions encountered on space missions and could be useful to elucidate the adaptation mechanisms of microbes to the space environment. [ABSTRACT FROM AUTHOR]

Bacterial RNA degradosomes are multienzyme molecular machines that act as hubs for post-transcriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications, and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation. In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries, and their similarities with the eukaryotic mRNP granules. RNA degradosomes, molecular machines composed of at least one RNase and one RNA helicase, are major players in post-transcriptional regulation of gene expression in bacteria. RNA degradosomes are diverse in composition and are tightly regulated by a wealth of mechanisms, including autoregulation, post-translational modifications, protein localization, or interaction with different partners. Some RNA degradosomes seem to be compartmentalized at the membrane, which could affect their activity. Some bacterial RNA degradosomes show similarities with eukaryotic mRNP granules at the level of their physicochemical properties. [ABSTRACT FROM AUTHOR]

Dual transcriptional profiling of host and bacteria during infection is challenging due to the low abundance of bacterial mRNA. We report Pathogen Hybrid Capture (PatH-Cap), a method to enrich for bacterial mRNA and deplete bacterial rRNA simultaneously from dual RNA-seq libraries using transcriptome-specific probes. By addressing both the differential RNA content of the host relative to the infecting bacterium and the overwhelming abundance of uninformative structural RNAs (rRNA, tRNA) of both species in a single step, this approach enables analysis of very low-input RNA samples. By sequencing libraries before (pre-PatH-Cap) and after (post-PatH-Cap) enrichment, we achieve dual transcriptional profiling of host and bacteria, respectively, from the same sample. Importantly, enrichment preserves relative transcript abundance and increases the number of unique bacterial transcripts per gene in post-PatH-Cap libraries compared to pre-PatH-Cap libraries at the same sequencing depth, thereby decreasing the sequencing depth required to fully capture the transcriptional profile of the infecting bacteria. We demonstrate that PatH-Cap enables the study of low-input samples including single eukaryotic cells infected by 1–3 Pseudomonas aeruginosa bacteria and paired host-pathogen temporal gene expression analysis of Mycobacterium tuberculosis infecting macrophages. PatH-Cap can be applied to the study of a range of pathogens and microbial species, and more generally, to lowly-abundant species in mixed populations. [ABSTRACT FROM AUTHOR]

The expression of four genes of bacterial endo-β-l,3(4)-D-glucanases from B. pumilus Bg57 (VKPM В-13195), Paenibacillus polymyxa Bg23 (VKPM В-4056), Bacillus subtilis Bg11 (VKPM B-13178), and Bacillus amyloliquefaciens Bg76 (VKPM B-13184), which are members of the family of GH16 glycohydrolases, in the expression system of Pichia pastoris are compared. Technologically valuable characteristics of the recombinant β-glucanases, such as the specific activity, pH and thermal stability, temperature and pH optima, resistance to digestive enzymes, and substrate specificity, were studied. It was shown that the enzymes from B. pumilus and P. polymyxa have the highest biotechnological potential for the creation of β‑glucanase producers based on recombinant P. pastoris yeast strains for further use in fodder production. [ABSTRACT FROM AUTHOR]

The expression of four genes of bacterial endo-β-l,3(4)-D-glucanases from B. pumilus Bg57 (VKPM В-13195), Paenibacillus polymyxa Bg23 (VKPM В-4056), Bacillus subtilis Bg11 (VKPM B-13178), and Bacillus amyloliquefaciens Bg76 (VKPM B-13184), which are members of the family of GH16 glycohydrolases, in the expression system of Pichia pastoris are compared. Technologically valuable characteristics of the recombinant β-glucanases, such as the specific activity, pH and thermal stability, temperature and pH optima, resistance to digestive enzymes, and substrate specificity, were studied. It was shown that the enzymes from B. pumilus and P. polymyxa have the highest biotechnological potential for the creation of β‑glucanase producers based on recombinant P. pastoris yeast strains for further use in fodder production. [ABSTRACT FROM AUTHOR]

The widespread use of metals influenced many researchers to examine the relationship between heavy metal toxicity and bacterial resistance. In this study, we have inoculated heavy metal-contaminated soil from Janghang region of South Korea in the nickel-containing media (20 mM Ni2+) for the enrichment. Among dozens of the colonies acquired from the several transfers and serial dilutions with the same concentrations of Ni, the strain Ni-2 was chosen for further studies. The isolates were identified for their phylogenetic affiliations using 16S rRNA gene analysis. The strain Ni-2 was close to Cupriavidus metallidurans and was found to be resistant to antibiotics of vancomycin, erythromycin, chloramphenicol, ampicillin, gentamicin, streptomycin, and kanamycin by disk diffusion method. Of the isolated strains, Ni-2 was sequenced for the whole genome, since the Ni-resistance seemed to be better than the other strains. From the genome sequence we have found that there was a total of 89 metal-resistance-related genes including 11 Ni-resistance genes, 41 heavy metal (As, Cd, Zn, Hg, Cu, and Co)-resistance genes, 22 cation-efflux genes, 4 metal pumping ATPase genes, and 11 metal transporter genes. [ABSTRACT FROM AUTHOR]

The biological characteristics and genome-wide sequence analysis of highly efficient endophytic nitrogen-fixing bacteria Klebsiella variicola GN02 isolated from the roots of Pennisetum sinense Roxb. were studied by the combination of second-generation and third-generation sequencing techniques. The cell cultivation characteristics, microscopic morphological observation and infrared spectroscopic analysis of this GN02 strain were performed and its genome assembly, gene prediction and functional annotation, eggNOG/GO clustering analysis and co-linearity analysis were conducted through corresponding software. The results showed that the GN02 strain had the basic morphological characteristics of genus Klebsiella and it had the characteristic absorption peak of marker C-N group and the characteristic absorption peaks of carbonyl and amide groups in nitrogen-fixing bacteria. The GN02 genome size was 5,599,366 bp with GC content of 57.41%, 5,261 ORF, 25 rRNA, 87 tRNA, 125 other ncRNA, 7 CRISPR repeats, and 54 GIs. Gene function annotations indicate that there were a large number of genes closely related to cellular nitrogen metabolism and the genes with high abundance are associated with amino acid metabolism. The genome-wide sequence has been submitted to the GenBank database under the accession number of CP31061. The basic characteristics of GN02 genome are similar to those of other K. variicola. Several strains of K. variicola have good co-linearity. The GN02 strain has a complete nitrogen-fixing gene group and 57 nitrogen-fixing genes have been identified. [ABSTRACT FROM AUTHOR]

Metagenome from refinery wastewater treatment plant running under nitrogen stress was analyzed for mining of novel aromatic hydrocarbon-degrading bacteria. The sequence data were assembled using metaspade followed by binning using the Metabat tool to assemble genome; where coverage and depth were calculated using bowtie and samtools. The analysis picked a novel genome belonging to family Bradyrhizobiaceae, identified based on 16S rDNA gene which was supported by CheckM and Kraken analysis. Using RAST, the assembled genome showed the capabilities for nitrogen fixation with the utilization of multiple hydrocarbon substrates with 14 different types of oxygenases as mapped by Minpath. An additional genetic feature like genes for stress and resistance towards heavy metals and antibiotic suggested that the genome has gone through the rigorous process of adaptation. If such bacteria could be cultivated then it will open the broad window of bioremediation strategies under nitrogen stress environment. • Genome assembly of a novel nitrogen fixing bacteria from metagenome • Bacterial ability to utilize various aromatic hydrocarbons • Gene cluster for carbon fixation, nitrogen metabolism and urea utilization • Large pool of stress and resistance related genes in the genome [ABSTRACT FROM AUTHOR]

Small regulatory RNAs (sRNAs) are increasingly recognized as players in the complex regulatory networks governing bacterial gene expression. RsaE (synonym RoxS) is an sRNA that is highly conserved in bacteria of the Bacillales order. Recent analyses in Bacillus subtilis , Staphylococcus aureus and Staphylococcus epidermidis identified RsaE/RoxS as a potent riboregulator of central carbon metabolism and energy balance with many molecular RsaE/RoxS functions and targets being shared across species. Similarities and species-specific differences in cellular processes modulated by RsaE/RoxS suggest that this sRNA plays a prominent role in the adaptation of Gram‐positive bacteria to niches with varying nutrient availabilities and environmental cues. This review summarizes recent findings on the molecular function of RsaE/RoxS and its interaction with mRNA targets. Special emphasis will be on the integration of RsaE/RoxS into metabolic regulatory circuits and, derived from this, the role of RsaE/RoxS as a putative driver to generate phenotypic heterogeneity in bacterial populations. In this respect, we will particularly discuss heterogeneous RsaE expression in S. epidermidis biofilms and its possible contribution to metabolic niche diversification, programmed bacterial lysis and biofilm matrix production. Unlabelled Image • RsaE (synonym RoxS) is a conserved sRNA of Gram-positive bacteria of the Bacillales order in which it exerts a profound influence on central carbon metabolism, energy balance and a multitude of other cellular functions. • Transcription of RsaE/RoxS is sensitive to the redox status of the cell and controlled via regulatory circuits involving transcription factors and efficient feedback loops, potentially favoring bistable expression of RsaE/RoxS within bacterial populations. • Heterogeneous RsaE/RoxS expression within S. epidermidis biofilms may contribute to metabolic niche diversification and facilitates biofilm matrix production through metabolic reprogramming as well as by mediating bacterial lysis and eDNA release. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a lifethreatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interactions between the host and bacteria have effects on bacterial gene expression profiles, while the gene expression pattern of S. pyogenes related to this disease remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by isolating total RNA from infected hind limbs obtained at 24, 48, and 96 h postinfection. RNA-seq analysis results identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs compared to their expression under in vitro conditions. Genes showing consistent enrichment during infection included 306 encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport, and the vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the present mouse model (log2 fold change, >6.0, >9.4, and >7.1, respectively). Conversely, the number of consistently downregulated genes was 177, including those associated with the oxidative stress response and cell division. These results suggest that in necrotizing fasciitis, S. pyogenes shows an altered metabolism, decreased cell proliferation, and upregulation of expression of major toxins. Our findings are considered to provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis. [ABSTRACT FROM AUTHOR]

It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA β-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection. [ABSTRACT FROM AUTHOR]

Background: Toxigenic Vibrio cholerae resides in aquatic reservoirs of cholera-endemic areas mostly in a dormant form known as conditionally viable environmental cells (CVEC) in which the bacteria remain embedded in an exopolysaccharide matrix, and fail to grow in routine bacteriological culture. The CVEC can be resuscitated by supplementing culture media with either of two autoinducers CAI-1 and AI-2, which are signal molecules controlling quorum sensing, a regulatory network of bacterial gene expression dependent on cell density. This study investigated possible existence of variant strains that overproduce AIs, sufficient to resuscitate CVEC in environmental waters. Methods: Environmental V. cholerae isolates and Tn insertion mutants of a V. cholerae strain C6706 were screened for production of AIs using bioluminescent reporter strains. Relevant mutations in environmental strains which overproduced AI-2 were characterized by nucleotide sequencing and genetic complementation studies. Effect of AIs produced in culture supernatants of relevant strains on reactivation of CVEC in water was determined by resuscitation assays. Results: Two of 54 environmental V. cholerae isolates were found to overproduce AI-2. Screening of a Tn-insertion library of V. cholerae strain C6706, identified a mutant which overproduced AI-2, and carried Tn insertion in the cqsS gene. Nucleotide sequencing also revealed mutations inactivating the cqsS gene in environmental isolates which overproduced AI-2, and this property was reversed when complemented with a wild type cqsS gene. Culture of river water samples supplemented with spent medium of these mutants resuscitated dormant V. cholerae cells in water. Significance: V. cholerae strains with inactivated cqsS gene may offer a convenient source of AI-2 in enhanced assays for monitoring bacteriological quality of water. The results also suggest a potential role of naturally occurring cqsS mutants in the environmental biology of V. cholerae. Furthermore, similar phenomenon may have relevance in the ecology of other waterborne bacterial pathogens beyond V. cholerae. [ABSTRACT FROM AUTHOR]

The objective of this study was to explore the effects of starter feeding on caecal mucosal bacterial composition and the expression of genes involved in immune and tight junctions in pre-weaned lambs. Six pairs of new-born twin lambs were selected. From 10 days of age, one lamb of each pair received ewe's milk only (M group, n = 6), while the other one was fed ewe's milk plus starter feed (M + S group, n = 6). At 56 days of age, the lambs were sacrificed, and then cecum digesta was collected to measure pH values and concentrations of volatile fatty acid (VFA), and caecal mucosa were collected to determine the changes in bacterial communities and the mRNA expression of cytokines, toll-like receptors (TLRs) and tight junction proteins. The results showed the body weight and average daily gain were not significantly different between both groups. Starter feeding significantly (P < 0.05) increased the concentrations of propionate and butyrate; the proportions of acetate, propionate and butyrate to total concentrations of VFA; and decreased the ratio of acetate to propionate in caecal contents. Principal coordinate analysis showed that samples from the M + S group could be distinguished from those from the M group; starter feeding also increased the diversity of caecal mucosal bacteria. At the genus level, starter feeding significantly (FDR < 0.05) increased the relative abundance of Alistipes , Parabacteroides , Parasutterella and Butyricimonas , and caused a decreasing trend (FDR < 0.10) in the relative abundance of Campylobacter and Helicobacter. The real-time PCR results showed that starter feeding significantly (FDR < 0.05) decreased the relative mRNA expression level of IL-12, TNF-α and TLR4 and increased the relative mRNA expression level of claudin-4. These results indicate that starter feeding altered caecal mucosal bacterial communities and decreased the expression of inflammatory factors, which may be beneficial in alleviating the weaning stress of lambs. • Starter feeding enhanced the caecal fermentation, and increased the bacterial diversity of caecal mucosa in lambs. • Starter feeding reduced the abundance of potentially harmful mucosa[HYPHEN]attached bacterial species in cecum of pre-weaned lambs. • Starter feeding increased the abundance of beneficial mucosa-attached bacterial species in cecum of pre-weaned lambs. • Starter feeding reduced the abundance of potentially harmful mucosa[HYPHEN]attached bacterial species in cecum of pre-weaned lambs. [ABSTRACT FROM AUTHOR]

Sensory systems such as the olfactory system detect chemical stimuli and thereby determine the relationships between the animal and its surroundings. Olfaction is one of the most conserved and ancient sensory systems in vertebrates. The vertebrate olfactory epithelium is colonized by complex microbial communities, but microbial contribution to host olfactory gene expression remains unknown. In this study, we show that colonization of germ-free zebrafish and mice with microbiota leads to widespread transcriptional responses in olfactory organs as measured in bulk tissue transcriptomics and RT-qPCR. Germ-free zebrafish olfactory epithelium showed defects in pseudostratification; however, the size of the olfactory pit and the length of the cilia were not different from that of colonized zebrafish. One of the mechanisms by which microbiota control host transcriptional programs is by differential expression and activity of specific transcription factors (TFs). REST (RE1 silencing transcription factor, also called NRSF) is a zinc finger TF that binds to the conserved motif repressor element 1 found in the promoter regions of many neuronal genes with functions in neuronal development and differentiation. Colonized zebrafish and mice showed increased nasal expression of REST, and genes with reduced expression in colonized animals were strongly enriched in REST-binding motifs. Nasal commensal bacteria promoted in vitro differentiation of Odora cells by regulating the kinetics of REST expression. REST knockdown resulted in decreased Odora cell differentiation in vitro. Our results identify a conserved mechanism by which microbiota regulate vertebrate olfactory transcriptional programs and reveal a new role for REST in sensory organs. [ABSTRACT FROM AUTHOR]

Psychrophilic and psychrotolerant bacteria from permanently cold environments may be the most abundant extremophiles on Earth and yet little is known on how they cope with temperature stress. Real-time reverse transcription PCR (RT-qPCR) is a powerful technique that could shed light on this matter but it requires pre-validated reference genes for normalization of data to get accurate results. In this study, we assessed the expression stability of eight candidate genes for the psychrotolerant Antarctic isolate Pseudomonas sp. AU10 during exponential growth under 4 °C and 30 °C, and after a cold-shock. Using the software programs BestKeeper and geNorm we validated recA, ftsZ, 16S rRNA, and rpoD as reference genes and we suggested the combination of recA and ftsZ for qPCR data normalization. Our results provide a starting point for gene expression studies in Antarctic Pseudomonas concerning temperature-related physiology and also for the validation of reference genes in other cold-adapted bacterial species. [ABSTRACT FROM AUTHOR]

Quorum Sensing (QS) is a mechanism of bacterial intercellular communication that depends on their population density. QS activates the expression of hundreds of bacterial genes, many of which play an important role in plant-bacterial interactions. This review summarizes the latest achievements in the research of bacterial QS signaling in plant-microbial interactions. The review focuses specifically on the best-studied QS systems, i.e., those of gram-negative bacteria which involve N-acyl-homoserine lactones (AHL). The effects of AHL and AHL-producing bacteria on plant growth, development, and defense responses are considered. Signaling functions of plant metabolites which regulate QS-dependent behavior of associated microbiota via the activation or inhibition of bacterial QS systems are described. Possible degradation pathways of AHL in the rhizosphere are also considered. Practical applications of QS regulation for plant protection are discussed. [ABSTRACT FROM AUTHOR]

Abstract Device-associated infection is becoming a major threat to patient health associated with the extensive use of indwelling medical devices. Bacterial biofilms formed on the surfaces of indwelling devices act as physical and biochemical barriers to protect bacterial cells from being killed by antibiotics and host-immune mediators. Although ultrasound (US) alone cannot destroy mature biofilms, it can sensitize Pseudomonas aeruginosa (P. aeruginosa) to gentamicin and disrupt the expression of genes associated with bacterial metabolism, motility, antibiotic resistance, virulence and quorum sensing involved in biofilm formation. Furthermore, by evaluating levels of reactive oxygen species in neutrophils, we showed that US may promote neutrophil phagocytosis of bacteria. Therefore, in addition to preventing the formation of P. aeruginosa biofilms in an amplitude-dependent manner, US can effectively destroy mature biofilms in combination with gentamicin and neutrophils. These results may have great potential for the treatment of device-associated infections. [ABSTRACT FROM AUTHOR]

The taxonomic assignment of uncultured prokaryotes to known taxa is a major challenge in microbial systematics. This relies usually on the phylogenetic analysis of the ribosomal small subunit RNA or a few housekeeping genes. Recent works have disclosed ribosomal proteins as valuable markers for systematics and, due to the boom in complete genome sequencing, their use has become widespread. Yet, in the case of uncultured strains, for which complete genome sequences cannot be easily obtained, sequencing many markers is complicated and time consuming. Taking the advantage of the organization of ribosomal protein coding genes in large gene clusters, we amplified a 32 kb conserved region encompassing the spectinomycin (spc) operon using long range PCR from isolated and from uncultured nodular endophytic Frankia strains. The phylogenetic analysis of the 27 ribosomal protein genes contained in this region provided a robust phylogenetic tree consistent with phylogenies based on larger set of markers, indicating that this subset of ribosomal proteins contains enough phylogenetic signal to address systematic issues. This work shows that using long range PCR could break down the barrier preventing the use of ribosomal proteins as phylogenetic markers when complete genome sequences cannot be easily obtained. [ABSTRACT FROM AUTHOR]

N-terminal coding sequences (NCSs) of genes significantly influence gene expression at the translation level and are important for fine-tuning gene expression in bacteria, however, engineering NCSs to fine-tune metabolic pathways is challenging. Here, we developed a statistics-guided native and synthetic NCSs engineering approach to fine-tune gene expression in the industrially important microorganism Bacillus subtilis. This method is based on experimentally characterizing and statistically analyzing 96 rationally selected NCSs from B. subtilis endogenous genes. These NCSs exhibited a magnitude difference of greater than 4 orders in their ability to drive gene expression in 4 different dynamic patterns, including growth-coupled, growth-delayed, consistent expression, and inhibitory patterns. Synthetic and native NCSs were used to fine-tune expression of key enzymes, identified via pathway analysis and kinetic modeling, in the biosynthetic pathway of the useful nutraceutical N -acetylneuraminic acid (NeuAc). We observed a 3.21-fold improvement in NeuAc biosynthesis, indicating that NCSs can provide a synthetic biology toolbox to fine-tune gene expression for metabolic engineering. • N-terminal coding sequences (NCSs) from Bacillus subtilis exhibited broad dynamic range for gene expression regulation. • Synthetic NCSs were developed guided by statistical analysis, which expanded dynamic range of gene expression regulation. • Synthetic and native NCSs were used to fine-tune N -acetylneuraminic acid pathway aided by kinetic modeling. [ABSTRACT FROM AUTHOR]

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments (TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent. Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacin-resistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased (p < 0.05) under low-nutrient (0.5 mg/L total organic carbon (TOC)) relative to high-nutrient (500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria (cip_3 strain) was significantly lower (p < 0.05) in low-nutrient conditions, with 10 mg/L TOC ((8.01 ± 0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC ((48.89 ± 4.16)-fold). Moreover, rpoS gene expression in resistant bacteria ((1.36 ± 0.13)-fold) was significantly lower (p < 0.05) than that in the wild-type strain ((2.78 ± 0.29)-fold) under low-nutrient conditions (10 mg/L TOC), suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller (p < 0.05) in low-nutrient conditions (5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics. Unlabelled Image [ABSTRACT FROM AUTHOR]

• We tested the capability of the acute phase proteins AGP to interact with S.aureus. • The study was carried out on biofilm forming S.aureus field strains. • AGP disrupts formed biofilm, but cannot interfere with de novo biofilm formation. • AGP dysregulates mRNA expression of genes related to biofilm formation. Staphylococcus aureus biofilm-related infections are of clinical concern due to the capability of bacterial colonies to adapt to a hostile environment. The present study investigated the capability of the acute phase protein alpha 1 -acid glycoprotein (AGP) to a) disrupt already established S. aureus biofilm and b) interfere with the biofilm de novo production by using Microtiter Plate assay (MtP) on field strains isolated from infected quarters by assessing. The present study also investigated whether AGP could interfere with the expression of bacterial genes related to biofilm formation (icaA, icaD, icaB, and icaC) and adhesive virulence determinants (fnbA, fnbB, clfA, clfB, fib, ebps, eno) by quantitative real-time PCR (qPCR). The results provided the evidence that AGP could disrupt the biofilm structure only when it was already developed, but could not prevent the de novo biofilm formation. Moreover, AGP could interfere with the expression levels of genes involved in biofilm formation in a dose- and strain-dependent way, by upregulating, or downregulating, icaABC genes and fnbB, respectively. The results presented in this study provide new insights about the direct antibacterial activity of AGP in bovine milk. It remains to be demonstrated the molecular bases of AGP mechanism of action, in particular for what concerns the scarce capability to interact with the de novo formation of biofilm. [ABSTRACT FROM AUTHOR]

• Bacteria communicate with plants by means of chemical signaling. • Quorum-sensing enables bacteria to synchronize expression of genes and behavior, which are critical for phytostimulation. • N -acyl- L -homoserine lactones and cyclodipeptides are perceived by plants to modulate growth and defense. • N -acyl- L -homoserine lactones may act as alarm signals, pathogen and/or microbe-associated molecular patterns. • Cyclodipeptides may function as hormonal mimics for plants. Bacteria rely on chemical communication to sense the environment and to retrieve information on their population densities. Accordingly, a vast repertoire of molecules is released, which synchronizes expression of genes, coordinates behavior through a process termed quorum-sensing (QS), and determines the relationships with eukaryotic species. Already identified QS molecules from Gram negative bacteria can be grouped into two main classes, N -acyl- L -homoserine lactones (AHLs) and cyclodipeptides (CDPs), with roles in biofilm formation, bacterial virulence or symbiotic interactions. Noteworthy, plants detect each of these molecules, change their own gene expression programs, re-configurate root architecture, and activate defense responses, improving in this manner their adaptation to natural and agricultural ecosystems. AHLs may act as alarm signals, pathogen and/or microbe-associated molecular patterns, whereas CDPs function as hormonal mimics for plants via their putative interactions with the auxin receptor Transport Inhibitor Response1 (TIR1). A major challenge is to identify the molecular pathways of QS-mediated crosstalk and the plant receptors and interacting proteins for AHLs, CDPs and related signals. [ABSTRACT FROM AUTHOR]