Deletion Mutant Strain Research Articles

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

The elucidation of the biodegradation of nitrobenzene and p-nitrophenol of nitroreductase from Antarctic psychrophile Psychrobacter sp. ANT206 under low temperature

Psychrobacter is one important typical strain in the Antarctic environment. In our previous study, Psychrobacter sp. ANT206 from Antarctica with novel cold-adapted nitroreductase (PsNTR) could biodegrade nitrobenzene and p-nitrophenol in low temperature environment. In this study, the in-frame deletion mutant of psntr (Δpsntr-ANT206) that displayed well genetic stability and kanamycin resistance stability was constructed using allelic replacement method. Additionally, Δpsntr-ANT206 was more sensitive to nitrobenzene and p-nitrophenol in the comparison of heat and hyperosmolarity, suggesting that psntr gene participated in the regulation of the tolerance against nitro-aromatic compounds (NACs). Further analysis was conducted by integrated gas chromatography-mass spectrometry (GC-MS), and several metabolites were identified. Among them, ethylbenzene, L-Alanine, citric acid, aniline, 4-aminophenol and other metabolites were different between the wild-type strain and Δpsntr-ANT206 under nitrobenzene and p-nitrophenol stress at different time periods under low temperature, respectively. These data could increase the knowledge of the construction of deletion mutant strains and biodegradation mechanism of NACs of typical strains Psychrobacter from Antarctica, which would also provide the basis of the molecular technique on the regulation of bioremediation of the contaminants under low temperature in the future.

ITRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.

Integrated Proteomic and Transcriptomic Analyses Reveal the Roles of Brucella Homolog of BAX Inhibitor 1 in Cell Division and Membrane Homeostasis of Brucella suis S2.

BAX inhibitor 1 (BI-1) is an evolutionarily conserved transmembrane protein first identified in a screening process for human proteins that suppress BAX-induced apoptosis in yeast cells. Eukaryotic BI-1 is a cytoprotective protein that suppresses cell death induced by multiple stimuli in eukaryotes. Brucella, the causative agent of brucellosis that threatens public health and animal husbandry, contains a conserved gene that encodes BI-1-like protein. To explore the role of the Brucella homolog of BI-1, BrBI, in Brucella suis S2, we constructed the brbI deletion mutant strain and its complemented strain. brbI deletion altered the membrane properties of Brucella suis S2 and decreased its resistance to acidic pH, H2O2, polymyxin B, and lincomycin. Additionally, deleting brbI led to defective growth, cell division, and viability in Brucella suis S2. We then revealed the effect of brbI deletion on the physiological characteristics of Brucella suis S2 via integrated transcriptomic and proteomic analyses. The integrated analysis showed that brbI deletion significantly affected the expression of multiple genes at the mRNA and/or protein levels. Specifically, the affected divisome proteins, FtsB, FtsI, FtsL, and FtsQ, may be the molecular basis of the impaired cell division of the brbI mutant strain, and the extensively affected membrane proteins and transporter-associated proteins were consistent with the phenotype of the membrane properties’ alterations of the brbI mutant strain. In conclusion, our results revealed that BrBI is a bacterial cytoprotective protein involved in membrane homeostasis, cell division, and stress resistance in Brucella suis S2.

Metabolomics and lipidomics analyses delineating Hfq deletion- induced metabolic alterations in Vibrio alginolyticus

Vibrio alginolyticus, a Gram-negative halophilic zoonotic pathogen, can cause bacterial hemorrhagic septicemia in various aquaculture species and wound infections in humans. V. alginolyticus brings about huge economic losses in aquaculture all around the world. Hfq, a ubiquitous RNA-binding protein, is a global post-transcriptional regulator in most bacteria and some archaea, by facilitating base-pairing between sRNAs and their mRNA targets, and is essential for adaptation to different environments and growth conditions. Despite its important roles in the stress adaptation, virulence, nutrient utilization, and colony morphology in V. alginolyticus, further research is required to shed light on the pathways modulated by Hfq. In this study, we used metabolomics and lipidomics analytical approaches based on ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to understand the impact of Hfq on the metabolic profile of V. alginolyticus. The deletion of the hfq gene resulted in the growth deficiency and cell elongation of V. alginolyticus cultured in the rich medium. Substantial metabolic alterations, mainly involved in amino acids, nucleotides, fatty acids, and lipids, were observed in the hfq deletion mutant strain (Δhfq) when compared to the wild-type (WT) strain and its complemented strain (hfq+). Notably, the hfq deletion induced accumulations of free fatty acids (FFAs) and nucleotides, but decreased levels of most amino acids (e.g., branched amino acids, aromatic amino acids, and aspartate family amino acids). Meanwhile, the majority of lipids (e.g., lysophosphatidylglycerol, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidic acid, cardiolipin, sphingomyelin, some phosphatidylglycerol and phosphatidylethanolamine with mono- or poly-unsaturated fatty acids) were decreased, while ceramide, diacylglycerol, triacylglycerol and some phosphatidylglycerol and phosphatidylethanolamine with saturated fatty acids increased in the Δhfq strain. These data demonstrated that the hfq deletion triggered de novo synthesis of FFAs and nucleosides and enhanced the catabolism of phospholipid and sphingomyelin to further regulate cell division and growth. Additionally, the hfq deletion could inhibit the synthesis of the branched amino acids, aromatic amino acids and enhance the TCA cycle to influence cell stress defenses and energy metabolism. Our research indicates that Hfq can regulate pathways associated with cell division, growth and stress resistance, being an important metabolic modulator in V. alginolyticus.

Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis.

Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His6-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD+, and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD+. The Km and kcat values were 131 ± 13 μM and 65 ± 5 min−1, respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway.

Integrated Transcriptomic and Proteomic Analyses Reveal the Role of NprR in Bacillus anthracis Extracellular Protease Expression Regulation and Oxidative Stress Responses.

NprR is a protein of Bacillus anthracis that exhibits moonlighting functions as either a phosphatase or a neutral protease regulator that belongs to the RNPP family. We previously observed that the extracellular protease activity of an nprR deletion mutant significantly decreased within in vitro cultures. To identify the genes within the regulatory network of nprR that contribute to its protease activity, integrated transcriptomic and proteomic analyses were conducted here by comparing the nprR deletion mutant and parent strains. A total of 366 differentially expressed genes (DEGs) between the strains were observed via RNA-seq analysis. In addition, label-free LC-MS/MS analysis revealed 503 differentially expressed proteins (DEPs) within the intracellular protein fraction and 213 extracellular DEPs with significant expressional differences between the strains. The majority of DEGs and DEPs were involved in environmental information processing and metabolism. Integrated transcriptomic and proteomic analyses indicated that oxidation-reduction-related GO terms for intracellular DEPs and endopeptidase-related GO terms for extracellular DEPs were significantly enriched in the mutant strain. Notably, many genes involved in protease activity were largely downregulated in the nprR deletion mutant cultures. Moreover, western blot analysis revealed that the major extracellular neutral protease Npr599 was barely expressed in the nprR deletion mutant strain. The mutant also exhibited impaired degradation of protective antigen, which is a major B. anthracis toxin component, thereby resulting in higher protein yields. Concomitantly, another global transcriptional regulator, SpxA1, was also dramatically downregulated in the nprR deletion mutant, resulting in higher sensitivity to oxidative and disulfide stress. These data consequently indicate that NprR is a transcriptional regulator that controls genes whose products function as extracellular proteases and also is involved in oxidative stress responses. This study thus contributes to a more comprehensive understanding of the biological function of NprR, and especially in the middle growth stages of B. anthracis.

6S-1 RNA Contributes to Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis.

6S RNA is a kind of high-abundance non-coding RNA that globally regulates bacterial transcription by interacting with RNA polymerase holoenzyme. Through bioinformatics analysis, we found that there are two tandem 6S RNA-encoding genes in the genomes of Bacillus cereus group bacteria. Using Bacillus thuringiensis BMB171 as the starting strain, we have explored the physiological functions of 6S RNAs, and found that the genes ssrSA and ssrSB encoding 6S-1 and 6S-2 RNAs were located in the same operon and are co-transcribed as a precursor that might be processed by specific ribonucleases to form mature 6S-1 and 6S-2 RNAs. We also constructed two single-gene deletion mutant strains ΔssrSA and ΔssrSB and a double-gene deletion mutant strain ΔssrSAB by means of the markerless gene knockout method. Our data show that deletion of 6S-1 RNA inhibited the growth of B. thuringiensis in the stationary phase, leading to lysis of some bacterial cells. Furthermore, deletion of 6S-1 RNA also significantly reduced the spore number and parasporal crystal content. Our work reveals that B. thuringiensis 6S RNA played an important regulatory role in ensuring the sporulation and parasporal crystal formation.

Involvement of a Novel TetR-Like Regulator (BdtR) of Bradyrhizobium diazoefficiens in the Efflux of Isoflavonoid Genistein

A wide variety of leguminous plant-released (iso)flavonoids, such as genistein, are potential inducers of the nodulation (nod) genes of endosymbiotic rhizobia for the production of Nod factors, which are vital signaling molecules for triggering the symbiotic process. However, these (iso)flavonoids are generally thought to be toxic to the bacterial partner to varying degrees. Here, a novel TetR-like regulator gene of the soybean symbiont Bradyrhizobium diazoefficiens USDA110, bdtR (systematic designation blr7023), was characterized. It was found to be rapidly and preferentially induced by genistein, and its mutation resulted in significantly increased expression of the neighboring bll7019-bll7021 genes, encoding a multidrug resistance efflux pump system, in the absence of this isoflavonoid. Then, the transcriptional start site of BdtR was determined, and it was revealed that BdtR acted as a transcriptional repressor of the above efflux system through the binding of an AT-rich operator, which could be completely prevented by genistein. In addition, the ΔbdtR deletion mutant strain showed higher accumulation of extracellular genistein and became less susceptible to the isoflavonoid. In contrast, the inactivation of BdtR led to the significantly decreased induction of a nodulation gene (nodY) independent of the expression of nodD1 and nodW and to much weaker nodulation competitiveness. Taken together, the results show that BdtR plays an early sensing role in maintaining the intracellular homeostasis of genistein, helping to alleviate its toxic effect on this bacterium by negatively regulating neighboring genes encoding an efflux pump system while being essentially required for nodule occupancy competitiveness.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dynamic transcriptional response of Saccharomyces cerevisiae cells to copper

Copper is a crucial trace element for all living systems and any deficiency in copper homeostasis leads to the development of severe diseases in humans. The observation of extensive evolutionary conservation in copper homeostatic systems between human and Saccharomyces cerevisiae made this organism a suitable model organism for elucidating molecular mechanisms of copper transport and homeostasis. In this study, the dynamic transcriptional response of both the reference strain and homozygous deletion mutant strain of CCC2, which encodes a Cu2+-transporting P-type ATPase, were investigated following the introduction of copper impulse to reach a copper concentration which was shown to improve the respiration capacity of CCC2 deletion mutants. The analysis of data by using different clustering algorithms revealed significantly affected processes and pathways in response to a switch from copper deficient environment to elevated copper levels. Sulfur compound, methionine and cysteine biosynthetic processes were identified as significantly affected processes for the first time in this study. Stress response, cellular response to DNA damage, iron ion homeostasis, ubiquitin dependent proteolysis, autophagy and regulation of macroautophagy, DNA repair and replication, as well as organization of mitochondrial respiratory chain complex IV, mitochondrial organization and translation were identified as significantly affected processes in only CCC2 deleted strain. The integration of the transcriptomic data with regulome revealed the differences in the extensive re-wiring of dynamic transcriptional organization and regulation in these strains.

Specific gene SEN1393 contributes to higher survivability of Salmonella Enteritidis in egg white by regulating sulfate assimilation pathway

Salmonella enterica serovar Enteritidis (S. Enteritidis) presents an excellent capacity to survive in egg white, which is a hostile environment for bacterial growth. To reveal its survival mechanism, this study focuses on the specific gene SEN1393, which has been found to exist only in the genomic sequence of S. Enteritidis. The survival capacity of the deletion mutant strain ΔSEN1393 was proven to be significantly reduced after incubation in egg white. RNA sequencing and RT-qPCR results demonstrate that the expression levels of 19 genes were up-regulated, while the expression levels of 9 genes were down-regulated in egg white. These genes were classified into 6 groups based on their functional categories, namely the sulfate assimilation pathway, arginine biosynthesis, the tricarboxylic acid cycle, the fimbrial protein, the transport and chelation of metal ion, and others (sctT, rhs, and pspG). The strain ΔSEN1393 was deduced to damage FeS cluster enzymes and increase the sulfate and iron requirements, and to reduce bacterial motility and copper homeostasis. Via InterProScan analysis, the gene SEN1393 was speculated to encode a TerB-like and/or DjlA-like protein, and therefore, together with cysJ, possibly reduced the oxidative toxicities resulting from oxyanions such as tellurite, and/or improved CysPUWA conformation to restrain the uptake of the toxic oxyanions. In summary, the gene SEN1393 enabled the higher survival of S. Enteritidis in egg white as compared to other pathogens by regulating the sulfate assimilation pathway.

Functional characterization of the catalytic and bromodomain of FgGCN5 in development, DON production and virulence of Fusarium graminearum

FgGCN5, a GCN5 homolog in Fusarium graminearum, plays a critical role in hyphal vegetative growth, asexual and sexual reproduction, deoxynivalenol (DON) biosynthesis and plant infection. For nuclear localized GCN5, four conserved sequence motifs (I–IV) are presented in the catalytic domain and a bromodomain in the carboxy-terminus. As a lysine acetyltransferase, conserved negatively charged residues are present to neutralize the protons from lysine substrates. However, the role of conserved motifs/domains and residues in FgGCN5 are unclear. Here, we generated deletion mutant strains for each the conserved motifs/domains and a glutamate residue 130 (E130) replacement mutant. Deletion of each conserved motif in the catalytic domain and replacement of E130 site resulted in manifold defects in hyphae growth, asexual and sexual development, DON biosynthesis, and plant infection. Phenotypic defects in the mutant strains were similar to deletion mutants. The deletion of the bromodomain led a significant reduction in DON production and virulence, with no effects on hyphae growth, asexual or sexual reproduction. FgGCN5 was further found to localize to the nucleus in conidia and hyphae cells. In conclusion, FgGCN5 encodes a nuclear localized acetyltransferase. The conserved motifs in the catalytic domain and E130 are essential for correct functions of the gene. The conserved bromodomain is important for DON production and pathogen virulence. This was the first report to identify the functions of conserved motifs/domains in FgGCN5, which will contribute to our understanding of the mechanism(s) by which FgGCN5 regulates F. graminearum.

Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of β-glucosidase function in Cellvibrio japonicus.

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates.

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

BackgroundMicrobial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.ResultsThis study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.ConclusionsApart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study

Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)2] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr)2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr)2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr)2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr)2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr)2 cytotoxicity.

Identification and characterization of a protein Bro1 essential for sophorolipids synthesis in Starmerella bombicola.

Sophorolipids (SLs) are surface-active molecules produced by the non-pathogenic yeast Starmerella bombicola CGMCC 1576. Several genes involved in the synthesis of SLs have been identified. However, the regulation mechanism of the synthesis pathway for SLs has not been investigated. We recently discovered a protein in S. bombicola, which is structurally related to Yarrowia lipolytica YlBro1. To identify the function of the protein SbBro1 in S. bombicola, the deletion, overexpression, and complementary mutant strains were constructed. We found that the deletion mutant no longer produced SLs. Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the Δbro1, especially the expression level of cyp52m1 encoding the first rate-limiting enzyme in SL synthesis pathway was down-regulated 13-folds and the expression of fatty acid β-oxidation-related enzymes was also down-regulated. This study can give insight into the regulation of SL synthesis.

Role of the ClpX from Corynebacterium crenatum involved in stress responses and energy metabolism.

ClpX and ClpP are involved in many important functions, including stress responses and energy metabolism, in microorganisms. However, the ClpX and ClpP of microbes used in industrial scale have rarely been studied. Industrial bacterial fermentation experiences a variety of stresses, and energy metabolism is extremely important for industrial bacteria. Thus, the role played by the ClpX and ClpP of industrial bacteria in fermentation should be investigated. Most microorganisms have a single clpP gene, while Corynebacterium crenatum AS 1.542 possesses two clpPs. Herein, the clpX, clpP1, and clpP2 of C. crenatum were cloned, and its fusion protein was expressed and characterized. We also constructed clpX deletion mutant and complementation strain. Results indicate that ClpX serves an important function in thermal, pH, and ethanol stresses. It is also involved in NADPH synthesis and glucose consumption during fermentation.

Homeobox proteins are essential for fungal differentiation and secondary metabolism in Aspergillus nidulans

The homeobox domain-containing transcription factors play an important role in the growth, development, and secondary metabolism in fungi and other eukaryotes. In this study, we characterized the roles of the genes coding for homeobox-type proteins in the model organism Aspergillus nidulans. To examine their roles in A. nidulans, the deletion mutant strains for each gene coding for homeobox-type protein were generated, and their phenotypes were examined. Phenotypic analyses revealed that two homeobox proteins, HbxA and HbxB, were required for conidia production. Deletion of hbxA caused abnormal conidiophore production, decreased the number of conidia in both light and dark conditions, and decreased the size of cleistothecia structures. Overexpressing hbxA enhanced the production of asexual spores and formation of conidiophore under the liquid submerged conditions. The hbxB deletion mutant strains exhibited decreased asexual spore production but increased cleistothecia production. The absence of hbxB decreased the trehalose content in asexual spores and increased their sensitivity against thermal and oxidative stresses. The ΔhbxA strains produced more sterigmatocystin, which was decreased in the ΔhbxB strain. Overall, our results show that HbxA and HbxB play crucial roles in the differentiation and secondary metabolism of the fungus A. nidulans.

Polymorphisms in RocA contribute to the molecular pathogenesis of serotype M28 group A streptococcus

Two‐component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine‐tune their activity. In group A streptococcus (GAS), RocA (regulator of Cov) is an accessory protein that functions with the CovRS (control of virulence regulator/sensor) TCS. Previous data suggest that the N‐terminal predicted transmembrane domains are important for RocA function and interaction with the CovRS TCS. Whole‐genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid altering) and nonsense (protein truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally‐occurring clinical isolates with rocA polymorphisms, an isogenic rocA deletion mutant strain, and isogenic rocA polymorphism mutant strains to perform genome‐wide transcript analysis (RNA‐seq), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change causing a wild‐type‐like virulence phenotype, or a substantial transcriptome change leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Additionally, in contrast to previously published data on functional domains of RocA, polymorphisms in both the N‐terminal transmembrane domains and the C‐terminal predicted non‐functional histidine kinase ATPase domain result in an altered virulence phenotype suggestive of loss of RocA activity. Taken together, our data show that naturally‐occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.Support or Funding InformationFondren Foundation, Houston Methodist Hospital and Research Institute (to JMM and PEB)American Heart Association Predoctoral Fellowship award 19PRE34380820 (to PEB)NIH grants R21AI139369‐01A1 and R21AI146771‐01 (to JMM)

Involvement of three FliA-family sigma factors in the sporangium formation, spore dormancy and sporangium dehiscence in Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms sporangia, which open up and release zoospores in response to water. Here, we report a genetic and functional analysis of four FliA-family sigma factors, FliA1, FliA2, FliA3 and FliA4. Transcription of fliA1, fliA2 and fliA3 was directly activated by the global transcriptional activator TcrA during sporangium formation and dehiscence, while fliA4 was almost always transcribed at low levels. Gene disruption analysis showed that (a) deletion of fliA2 reduced the zoospore swimming speed by half, (b) the fliA1-fliA2 double-deletion mutant formed abnormal sporangia in which mutant spores ectopically germinated and (c) deletion of fliA3 induced no phenotypic changes in the wild-type and mutant strains of fliA1 and/or fliA2. Comparative RNA-Seq analyses among the wild-type and gene deletion mutant strains showed probable targets of each FliA-family sigma factor, indicating that FliA1- and FliA2-dependent promoters are quite similar to each other, while the FliA3-dependent promoter is somewhat different. Gene complementation experiments also indicated that the FliA1 regulon overlaps with the FliA2 regulon. These results demonstrate that A. missouriensis has developed a complex transcriptional regulatory network involving multiple FliA-family sigma factors for the accomplishment of its characteristic reproduction process, including sporangium formation, spore dormancy and sporangium dehiscence.