Gene Of Operon Research Articles

The plasmid-encoded lactose operon plays a vital role in the acid production rate of Lacticaseibacillus casei during milk beverage fermentation.

Lacticaseibacillus casei is used extensively in the fermented milk-beverage industry as a starter culture. Acid production capacity during fermentation is the main criterion for evaluating starters although it is strain-dependent. In this study, the acid production rates of 114 L. casei strains were determined and then classified into high acid (HC), medium acid (MC), and low acid (LC) groups. Comparative genomics analysis found that the lac operon genes encoding the phosphoenolpyruvate-lactose phosphotransferase system (PTSLac) were located on plasmids in the HC strains; however, it is notable that the corresponding operons were located on the chromosome in LC strains. Real-time PCR analysis showed that the copy numbers of lac operon genes in HC strains were between 3.1 and 9.3. To investigate the relationship between copy number and acid production rate, the lac operon cluster of the HC group was constitutively expressed in LC strains. The resulting copy numbers of lac operon genes were between 15.8 and 18.1; phospho-β-galactosidase activity increased by 1.68-1.99-fold; and the acid production rates increased by 1.24-1.40-fold, which enhanced the utilization rate of lactose from 17.5 to 42.6% in the recombinant strains. The markedly increased expression of lac operon genes increased lactose catabolism and thereby increased the acid production rate of L. casei.

Mechanism of biofilm formation by Salmonella typhimurium ST19 in a high-glucose environment revealed by transcriptomics

Salmonella is one of the most common foodborne pathogens in the world, and Salmonella typhimurium, the most frequently isolated serotype, can cause zoonosis. In this study, we evaluated the effect of glucose concentration on the biofilm formation of 11 strains of Salmonella typhimurium. We found that among the selected strains, low-sugar conditions were more conducive to the formation of biofilms, although strain ST19 followed another rule. As the glucose concentration increased, biofilm formation showed an increasing trend. When the concentration of glucose reached 1.5%, other strains no longer formed biofilms, while strain ST19 still had strong biofilm formation ability. Therefore, we selected a culture medium containing 1.5% glucose to collect biofilms and planktonic cells of strain ST19 and compared them with strain CDC3, which did not produce a biofilm under these conditions, for transcriptomics analysis. The results showed that the related genes in the phosphotransferase system (PTS) and the operon genes regulated by AI-2 signaling molecules were highly downregulated in the biofilm bacteria of strain ST19 with a glucose content of 1.5%. At the same time, the genes involved in the two-component system, carbon metabolism and transcription regulation were significantly upregulated, which indicated that strain ST19 could adapt to a high-glucose environment by regulating glucose transport and metabolism in bacteria under a high-glucose environment and regulate biofilm formation by upregulating the expression of related genes in the two-component system. These findings illustrated that the regulated mechanism of Salmonella typhimurium's biofilm formation, which are significant to control biofilm contamination.

Positive regulation of the DLT operon by TCSR7 enhances acid tolerance of Lactococcus lactis F44.

Lactococcus lactis, a lactic acid bacterium, has been widely used in the fermented dairy products. The acid tolerance of L. lactis is of great importance to food fermentation and probiotic applications. As the first barrier of bacteria, the cell wall has a protective effect on strains under many stress conditions, whereas the regulatory mechanism has rarely been reported. Here, based on the transcription analysis of 9 cell wall or membrane-related genes of L. lactis F44 under acid stress, the transcription levels of DACB, DLTD, YLBA, HRTA, WP_080613266.1 (1610), and ERFK genes were significantly increased. We constructed 9 overexpressing strains with the cell wall or membrane-related genes, respectively. It was demonstrated that the survival rates under acid stress of DACB, DLTD, and ERFK were significantly higher than that of wild-type F44. To investigate the regulatory mechanism, a DNA pull-down assay was used to identify the transcriptional regulators of these 3 genes. It was discovered that the 2-component system (TCS) transcriptional regulator TCSR7 bound to the upstream region of DLTD involved in the teichoic acid (TA) alanylation. The combination was confirmed through an electrophoretic mobility shift assay in vitro. Reverse-transcription quantitative PCR results indicated that TCSR7 upregulated the expression of DLTD gene. In addition, the transcription level of TCSR7 increased approximately 1.8-fold (log2 fold change) under acidic conditions. In summary, this study found that TCSR7 was induced by acid stress to upregulate the transcription level of the DLT operon genes, which might increase the positive charge on the cell membrane surface to increase the acid tolerance of the strain. This study lays the foundation for the regulatory mechanism of TA alanylation under acid stress.

Characterization and comparative transcriptome analyses of Salmonella enterica Enteritidis strains possessing different chlorine tolerance profiles

We compared viability, morphology, enzyme activity and gene expression patterns between the chlorine tolerant S. Enteritidis RE 13 and the sensitive strain S. Enteritidis CVCC1806. As expected, viability of strain CVCC1806 was significantly lower than for RE 13 after exposure to 100 mg/L NaClO for 20 min and the sensitive strain displayed severe cell membrane damage and intracellular ATP depletion compared with the tolerant strain. The NAD+/NADH ratios for the sensitive vs tolerant strain were also significantly (p < 0.05) different (3.59 ± 0.95 vs. 0.88 ± 0.47) and indicated that tolerance included adaptation to redox perturbations. Transcriptomes of these two strains following chlorine exposure identified significant up-regulation of efflux pump systems (proV/W, OmpF, marA), oxidative stress response (trxC, yhhP, soxS) and DNA repair (dnaQ, polB, yaiV) for the tolerant RE 13 strain. These data indicated that S. Enteritidis RE 13 has a more robust response to oxidative stress that acted to maintain its intracellular redox balance to adapt and survive in the presence of chlorine. Noteworthy, the operon gene cluster of the bacterial microcompartment PDU were up-regulated about 64-fold in tolerant strain RE 13 compared with the sensitive strain, posing a more serious threat to human intestinal health.

Complete genome sequences of two Escherichia coli clinical isolates from Egypt carrying mcr-1 on IncP and IncX4 plasmids.

Colistin is a last-resort antibiotic used in the treatment of multidrug resistant Gram-negative bacteria. However, the activity and efficacy of colistin has been compromised by the worldwide spread of the mobile colistin resistance genes (mcr-1 to mcr-10). In this study, two clinical Escherichia coli strains, named EcCAI51, and EcCAI73, harbored mcr-1, showed multidrug-resistant phenotypes (with colistin MIC = 4 μg/ml), and belonged to phylogroup D: multilocus sequence type 1011 (ST1011) and phylogroup A: ST744, respectively. Findings revealed the existence of mcr-1 gene on two conjugable plasmids, pAMS-51-MCR1 (∼122 kb IncP) and pAMS-73-MCR1 (∼33 kb IncX4), in EcCAI51, and EcCAI73, respectively. The mcr-1-pap2 element was detected in the two plasmids. Additionally, the composite transposon (ISApl1-IS5D-pap2-mcr-1-ISApl1) was identified only in pAMS-51-MCR1 suggesting the potential for horizontal gene transfer. The two strains carried from 16 to 18 different multiple acquired antimicrobial resistance genes (ARGs). Additionally, two different multireplicon virulence plasmids (∼117 kb pAMS-51-Vr and ∼226 kb pAMS-73-Vr) carrying the sit operon, the Salmochelin siderophore iroBCDE operon and other several virulence genes were identified from the two strains. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of EcCAI73, and EcCAI51 with global E. coli lineages at HC levels of 50 (HC50) to 100 (HC100) core genome allelic differences. To the best of our knowledge, this study presented the first complete genomic sequences of mcr-1-carrying IncP and IncX4 plasmids from human clinical E. coli isolates in Egypt. In addition, the study illustrated the mcr-1 broad dissemination in diverse plasmids and dissimilar E. coli clones.

Distribution of papG alleles among uropathogenic Escherichia coli from reproductive age women

BackgroundExtraintestinal Escherichia coli (E. coli) causing urinary tract infections (UTIs), and often referred to as uropathogenic E. coli (UPEC), are a major contributor to the morbidity of UTIs and associated healthcare costs. UPEC possess several virulence factors (VFs) for infecting and injuring the host. We studied the papG allele distribution, and its association with other VF genes and phylogenetic groups, amongst 836 UPEC and fecal isolates from reproductive age women.ResultsThe papGII gene was highly prevalent amongst pyelonephritis isolates (68%), whilst the majority, albeit smaller proportion, of cystitis isolates (31%) harboured the papGIII gene. Among the pyelonephritis and cystitis isolates, papG positive isolates on average had higher VF gene scores, and were more likely to belong to phylogenetic group B2, than their negative counterparts. This was mostly due to the contribution of papGII isolates, which on average contained more VF genes than their papGIII counterparts, irrespective of the uro-clinical syndrome. However, the papGII isolates from the pyelonephritis cohort had higher VF gene scores than the cystitis ones, suggesting presence of possible papGII clones with differing inferred virulence potential. Furthermore, papGII isolates were more likely to possess an intact pap gene operon than their papGIII counterparts. Also of note was the high proportion of isolates with the papGI allele which was not associated with other pap operon genes; and this finding has not been described before.ConclusionsThe association of the papGII gene with several VF genes compared to the papGIII gene, appears to explain the abundance of these genes in pyelonephritis and cystitis isolates, respectively.

Draft Genome Sequence of Multidrug Resistant Klebsiella pneumoniae Strain C43 Isolated from Environmental Water Sample.

ABSTRACTHere, we report the genome of ESBL-producing Klebsiella pneumoniae strain C43, which was isolated from an environmental water sample. The genome is 5,614,412 bp in size with GC content of 56.86% with multidrug antimicrobial resistance genes and several metal resistance gene operons.

In-silico analyses provide strong statistical evidence for intra-species recombination events of the gyrA and CmeABC operon loci contributing to the continued emergence of resistance to fluoroquinolones in natural populations of Campylobacter jejuni

The continued emergence of Campylobacter jejuni strains resistant to fluoroquinolones (FQs) has posed a significant threat to global public health, leading frequently to undesirable outcomes of human campylobacteriosis treatment. The molecular genetic mechanisms contributing to the increased retention of resistance to FQs in natural populations of this species, especially in antibiotic-free environments, are not clearly understood. This study aimed to determine whether genetic recombination could be such a mechanism. We applied a large array of algorithms, imbedded in the SplitsTree and RDP4 software packages, to analyse the DNA sequences of the chromosomal loci, including the gyrA gene and the CmeABC operon, to identify events of their genetic recombination between C. jejuni strains. The SplitsTree analyses of the above genetic loci resulted in several parallelograms with the bootstrap values being in a range of 94.7 to 100, with the high fit estimates being 99.3 to 100. These analyses were further strongly supported by the Phi test results (P ≤ 0.02715) and the RDP4-generated statistics (P ≤ 0.04005). The recombined chromosomal regions, along with the gyrA gene and CmeABC operon loci, were also found to contain the genetic loci that included, but were not limited to, the genes encoding for phosphoribosyltransferase, lipoprotein, outer membrane motility protein, and radical SAM domain protein. These findings strongly suggest that the genetic recombination of the chromosomal regions involving gyrA, CmeABC, and their adjacent loci may be an additional mechanism underlying the constant emergence of epidemiologically successful FQ-resistant strains in natural populations of C. jejuni.

In Silico Study of Mercury Resistance Genes Extracted from Pseudomonas spp. Involved in Bioremediation: Understanding the Promoter Regions and Regulatory Elements.

Microbial genes and their product were diverse and beneficial for heavy metal bioremediation from the contaminated sites. Screening of genes and gene products plays a significant role in the detoxification of pollutants. Understanding of the promoter region and its regulatory elements is a vital implication of microbial genes. To the best of our knowledge, there is no in silico study reported so far on mer gene families used for heavy metal bioremediation. The motif distribution was observed densely upstream of the TSSs (transcription start sites) between +1 and -350 bp and sparsely distributed above -350 bp, according to the current study. MEME identified the best common candidate motifs of TFs (transcription factors) binding with the lowest e value (7.2e-033) and is the most statistically significant candidate motif. The EXPREG output of the 11 TFs with varying degrees of function such as activation, repression, transcription, and dual purposes was thoroughly examined. Data revealed that transcriptional gene regulation in terms of activation and repression was observed at 36.4% and 54.56%, respectively. This shows that most TFs are involved in transcription gene repression rather than activation. Likewise, EXPREG output revealed that transcriptional conformational modes, such as monomers, dimers, tetramers, and other factors, were also analyzed. The data indicated that most of the transcriptional conformation mode was dual, which accounts for 96%. CpG island analysis using online and offline tools revealed that the gene body had fewer CpG islands compared to the promoter regions. Understanding the common candidate motifs, transcriptional factors, and regulatory elements of the mer operon gene cluster using a machine learning approach could help us better understand gene expression patterns in heavy metal bioremediation.

Evidence for further non-coding RNA genes in the fungal rDNA region.

Non-coding RNA (ncRNA) genes play important, but incompletely understood, roles in various cellular processes, notably translation and gene regulation. A recent report on the detection of the ncRNA Signal Recognition Particle gene in the nuclear ribosomal internal transcribed spacer region of several species of three genera of ectomycorrhizal basidiomycetes prompted a more thorough bioinformatics search for additional ncRNA genes in the full fungal ribosomal operon. This study reports on the detection of three ncRNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of Auricularia and Inocybe. We verified their presence through resequencing of independent specimens. Two completed Auricularia genomes were found to lack these ncRNAs elsewhere than in the ribosomal operon, suggesting that these are functional genes. It seems clear that ncRNA genes play a larger role in fungal ribosomal genetics than hitherto thought.

A hybrid type of chromatic acclimation regulated by the dual green/red photosensory systems in cyanobacteria.

Cyanobacteria are phototrophic bacteria that perform oxygenic photosynthesis. They use a supermolecular light-harvesting antenna complex, the phycobilisome (PBS), to capture and transfer light energy to photosynthetic reaction centers. Certain cyanobacteria alter the absorption maxima and/or overall structure of their PBSs in response to the ambient light wavelength-a process called chromatic acclimation (CA). One of the most well-known CA types is the response to green and red light, which is controlled by either the RcaEFC or CcaSR photosensory system. Here, we characterized a hybrid type of CA in the cyanobacterium Pleurocapsa sp. Pasteur Culture Collection (PCC) 7319 that uses both RcaEFC and CcaSR systems. In vivo spectroscopy suggested that strain PCC 7319 alters the relative composition of green-absorbing phycoerythrin and red-absorbing phycocyanin in the PBS. RNA sequencing and promoter motif analyses suggested that the RcaEFC system induces a gene operon for phycocyanin under red light, whereas the CcaSR system induces a rod-membrane linker gene under green light. Induction of the phycoerythrin genes under green light may be regulated through a yet unidentified photosensory system called the Cgi system. Spectroscopy analyses of the isolated PBSs suggested that hemidiscoidal and rod-shaped PBSs enriched with phycoerythrin were produced under green light, whereas only hemidiscoidal PBSs enriched with phycocyanin were produced under red light. PCC 7319 uses the RcaEFC and CcaSR systems to regulate absorption of green or red light (CA3) and the amount of rod-shaped PBSs (CA1), respectively. Cyanobacteria can thus flexibly combine diverse CA types to acclimate to different light environments.

Towards synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Staphylococcal saoABC Operon Codes for a DNA-Binding Protein SaoC Implicated in the Response to Nutrient Deficit.

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.

The carnitine degradation pathway of Acinetobacter baumannii and its role in virulence.

The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO2 and pyruvate and, that not, as previously suggested, l-malate is the product and funnelled directly into the TCA cycle. Mutant analyses revealed that the hydrolase in this cluster funnels acetylcarnitine into the degradation pathway by deacetylation. A transcriptional regulator CarR bound in a concentration-dependent manner to the intergenic region between the mdh gene, the first gene of the carnitine catabolic operon and the carR gene in the presence and absence of carnitine. Both carnitine and d-malate induced CarR-dependent expression of the carnitine operon. Infection studies with Galleria mellonella larvae demonstrated a strong increase in virulence by addition of carnitine indicating that carnitine degradation plays a pivotal role in virulence of A. baumannii.

Transcriptional profiles of Streptococcus pneumoniae associated with adaptation to the nasopharynx environment

Streptococcus pneumoniae is a major cause of global morbidity and mortality. It behaves as a commensal in the host nasopharynx, but can become pathogenic, invading the lungs, blood, and meninges. As such, identification of pneumococcal virulence and colonisation factors remains a major research objective. We previously described an experimental evolution approach for the identification of pneumococcal genes that make niche-specific contributions to fitness and virulence. Sequential passage of pneumococci through mouse models of nasopharyngeal-carriage and pneumonia was performed, generating bacterial lineages adapted to the nasopharynx and lungs, respectively. Using RNA-Seq differential gene expression analysis, this study compared the transcriptomic profile of a nasopharynx-evolved pneumococcal lineage that showed evidence of enhanced nasopharyngeal colonisation potential, with a lab-adapted ancestor strain. Here, we describe how the genomic adaptations acquired by this lineage, and which we have demonstrated facilitate survival in the nasopharynx, can influence bacterial gene expression. One key finding was the identification of five adjacent upregulated genes, representing a putative pneumococcal operon. These poorly characterised genes are predicted to encode a carbohydrate-scavenging pathway. Expression of the operon within the nasopharynx may facilitate sugar acquisition from host glycoproteins. Of note, the nasopharynx evolved pneumococcal lineage carries a single nucleotide insertion mutation immediately adjacent to the -10 element of the operon predicted promotor sequence. This may contribute to increased operon gene expression in the nasopharynx-adapted pneumococcus, thereby enhancing colonisation potential. Confirmatory mechanistic investigations are underway, which will aid the identification of pneumococcal virulence factors involved in the commensal to pathogen switch.

Characterization of qnrB-carrying plasmids from ESBL- and non-ESBL-producing Escherichia coli

BackgroundEscherichia coli carrying clinically important antimicrobial resistances [i.e., against extended-spectrum-beta-lactamases (ESBL)] are of high concern for human health and are increasingly detected worldwide. Worryingly, they are often identified as multidrug-resistant (MDR) isolates, frequently including resistances against quinolones/fluoroquinolones.ResultsHere, the occurrence and genetic basis of the fluoroquinolone resistance enhancing determinant qnrB in ESBL-/non-ESBL-producing E. coli was investigated. Overall, 33 qnrB-carrying isolates out of the annual German antimicrobial resistance (AMR) monitoring on commensal E. coli (incl. ESBL-/AmpC-producing E. coli) recovered from food and livestock between 2013 and 2018 were analysed in detail. Whole-genome sequencing, bioinformatics analyses and transferability evaluation was conducted to characterise the prevailing qnrB-associated plasmids. Furthermore, predominant qnrB-carrying plasmid-types were subjected to in silico genome reconstruction analysis. In general, the qnrB-carrying E. coli were found to be highly heterogenic in their multilocus sequence types (STs) and their phenotypic resistance profiles. Most of them appeared to be MDR and exhibited resistances against up to ten antimicrobials of different classes. With respect to qnrB-carrying plasmids, we found qnrB19 located on small Col440I plasmids to be most widespread among ESBL-producing E. coli from German livestock and food. This Col440I plasmid-type was found to be highly conserved by exhibiting qnrB19, a pspF operon and different genes of unassigned function. Furthermore, we detected plasmids of the incompatibility groups IncN and IncH as carriers of qnrB. All qnrB-carrying plasmids also exhibited virulence factors and various insertion sequences (IS). The majority of the qnrB-carrying plasmids were determined to be self-transmissible, indicating their possible contribution to the spread of resistances against (fluoro)quinolones and other antimicrobials.ConclusionIn this study, a diversity of different plasmid types carrying qnrB alone or in combination with other resistance determinants (i.e., beta-lactamase genes) were found. The spread of these plasmids, especially those carrying antimicrobial resistance genes against highest priority critically important antimicrobial agents, is highly unfavourable and can pose a threat for public health. Therefore, the dissemination pathways and evolution of these plasmids need to be further monitored.

Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Streptomyces Hosts.

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 μg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.

SdiA Enhanced the Drug Resistance of Cronobacter sakazakii and Suppressed Its Motility, Adhesion and Biofilm Formation.

Cronobacter sakazakii is a common foodborne pathogen, and the mortality rate of its infection is as high as 40–80%. SdiA acts as a quorum sensing regulator in many foodborne pathogens, but its role in C. sakazakii remains unclear. Here, we further determined the effect of the sdiA gene in C. sakazakii pathogenicity. The SdiA gene in C. sakazakii was knocked out by gene editing technology, and the biological characteristics of the ΔsdiA mutant of C. sakazakii were studied, followed by transcriptome analysis to elucidate its effects. The results suggested that SdiA gene enhanced the drug resistance of C. sakazakii but diminished its motility, adhesion and biofilm formation ability and had no effect on its growth. Transcriptome analysis showed that the ΔsdiA upregulated the expression levels of D-galactose operon genes (including dgoR, dgoK, dgoA, dgoD and dgoT) and flagella-related genes (FliA and FliC) in C. sakazakii and downregulated the expression levels of related genes in the type VI secretion system (VasK gene was downregulated by 1.53-fold) and ABC transport system (downregulated by 1.5-fold), indicating that SdiA gene was related to the physiological metabolism of C. sakazakii. The results were useful for clarifying the pathogenic mechanism of C. sakazakii and provide a theoretical basis for controlling bacterial infection.

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system.

Bacteriophage exclusion (‘BREX’) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR (‘BREX Regulator’), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

Microbial mercury methylation potential in a large-scale municipal solid waste landfill, China

Landfills harbor ideal conditions for microbial mercury methylation. However, the levels and distribution of mercury (Hg) and methylmercury (MeHg), potential microbial Hg methylation, and their linkage within landfills are largely unknown. In the present study, total mercury (THg), MeHg, the Hg methylation gene (hgcA) and mer operon were quantified in 30 waste samples from different depths (0–30 m) at 5 locations within a large-scale landfill in China. The average concentrations of THg and MeHg in the solid waste samples were 1422.91 ng/g and 3.15 ng/g, respectively. THg (up to 14405.29 ng/g) and MeHg (up to 10.42 ng/g) have high concentrations in the middle part (10–15 m) along the depth profiles. The concentration of THg was strongly positively (both p < 0.05) correlated with the MeHg concentration and the relative abundance of hgcA, indicating that the THg concentration can play an important role in microbial Hg methylation. The hgcA genes were detected in most samples and mer operon were detected in all samples. Combined hgcA qPCR and metagenomics data showed that Archaea Methanofollis may mainly account for Hg methylation within landfills. These findings provide fundamental knowledge on Hg cycles in landfills.