Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this ...study, we isolated a less-susceptible strain of
to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced
strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain
ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of
INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental
isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.
Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, ...such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. In this study, we analyze existing transcriptome data from a B. subtilis alpha-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2-threefold increase in alpha-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased alpha-amylase yield by about 30%. Transcript levels of the alpha-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. We demonstrate that the disruption of flagella in a B. subtilis alpha-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of alpha-amylase in small-scale fermentation.
Life emerged in an anoxic world, but the release of molecular oxygen, the by-product of photosynthesis, forced adaptive changes to counteract its toxicity. However, reactive oxygen species can damage ...all cellular components, including proteins. Therefore, several mechanisms have evolved to balance the intracellular redox state and maintain a reductive environment more compatible with many essential biological functions. In this study, we statistically interrogated the amino acid composition of
E. coli
proteins to investigate how the proneness or susceptibility to oxidation of amino acids biased their sequences. By sorting the proteins into five compartments (cytoplasm, internal membrane, periplasm, outer membrane, and extracellular), we found that various oxidative lesions constrain protein composition and depend on the cellular compartments, impacting the evenness of distribution or frequency. Our findings suggest that oxidative susceptibility could influence the observed differences in amino acid abundance across cellular compartments. This result reflects how the oxidative atmosphere could restrict protein amino acid composition and impose a codon bias trend.
Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair ...bacterial DNA damage induced by antimicrobials such as quinolones. The extent of
-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of
clinical strains.
Five clinical isolates of
carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with
ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the
gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of
in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software.
The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins.
High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.
CrAssphages represent the most abundant virus in the human gut microbiota, but the lack of available genome sequences for comparison has kept them enigmatic. Recently, sequence-based classification ...of distantly related crAss-like phages from multiple environments was reported, leading to a proposed familial-level taxonomic group. Here, we assembled the metagenomic sequencing reads from 702 human fecal virome/phageome samples and analyzed 99 complete circular crAss-like phage genomes and 150 contigs ≥70 kb. In silico comparative genomics and taxonomic analysis enabled a classification scheme of crAss-like phages from human fecal microbiomes into four candidate subfamilies composed of ten candidate genera. Laboratory analysis was performed on fecal samples from an individual harboring seven distinct crAss-like phages. We achieved crAss-like phage propagation in ex vivo human fecal fermentations and visualized short-tailed podoviruses by electron microscopy. Mass spectrometry of a crAss-like phage capsid protein could be linked to metagenomic sequencing data, confirming crAss-like phage structural annotations.
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•Screening of human fecal metagenomic samples reveals 249 crAss-like phage genomes•The crAss-like phages were classified into 4 subfamilies composed of 10 candidate genera•A crAss-like phage was propagated in ex vivo human fecal fermentations•Short-tailed phage virions could be visualized by electron microscopy
CrAssphage is the most abundant human gut-associated virus. Guerin et al. identify 249 crAss-like phage genomes and classify them into four subfamilies and ten candidate genera that differ among human populations. These in silico predictions are combined with ex vivo propagations, electron microscopy imaging, and mass spectrometry detection.
Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, but others are active and can be triggered ...into a replicative form either spontaneously or by exposure to inducing factors. Prophages are commonly associated with the ability to confer toxin production or other virulence-associated traits on their host cell. More recent studies have shown they can play a much bigger role in altering the physiology of their hosts. The technique described here has enabled us to investigate how prophages affect gene expression in the opportunistic bacterium Pseudomonas aeruginosa. In this work, the growth of the wild-type P. aeruginosa strain PAO1 was compared with that of isogenic lysogens carrying different combinations of prophages from the Liverpool Epidemic Strain (LES) LESB58. In a lysogen culture, a proportion of bacterial cells will be supporting lytic bacteriophage replication (spontaneous induction) with a high level of expression per cell of late phage genes, such as those associated with the assembly of phage particles, thus masking the low-level gene expression associated with lysogen-restricted gene expression. The impact of spontaneous induction can thus obscure prophage gene expression across a lysogen population. Growth profiling experiments were used to identify spontaneous induction, which was minimal during the early exponential growth phase. This study reports how to prepare sample cultures during the early exponential growth phase and how to set up adequate controls despite low cell numbers. These protocols ensure the reliable and reproducible comparison of wild-type and lysogenic bacteria under various conditions, thus improving the transcriptomic profiling of prophage genomes and aiding in the identification of previously unrecognized prophage functions.
•The Bacillaceae-1 RNA motif consists of two distinct classes.•Reverse complementary B-1 structure upstream of 16S rRNA has one stable motif.•Intergenic B-1 motifs have high structure diversity and ...may act as structural switch.
Non-coding RNAs are key regulatory players in bacteria. Many computationally predicted non-coding RNAs, however, lack functional associations. An example is the Bacillaceae-1 RNA motif, whose Rfam model consists of two hairpin loops. We find the motif conserved in nine of 13 non-pathogenic strains of the genus Bacillus but only in one pathogenic strain. To elucidate functional characteristics, we studied 118 hits of the Rfam model in 11 Bacillus spp. and found two distinct classes based on the ensemble diversity of their RNA secondary structure and the genomic context concerning the ribosomal RNA (rRNA) cluster. Forty hits are associated with the rRNA cluster, of which all 19 hits upstream flanking of 16S rRNA have a reverse complementary structure of low structural diversity. Fifty-two hits have large ensemble diversity, of which 38 are located between two coding genes. For eight hits in Bacillus subtilis, we investigated public expression data under various conditions and observed either the forward or the reverse complementary motif expressed. Five hits are associated with the rRNA cluster. Four of them are located upstream of the 16S rRNA and are not transcriptionally active, but instead, their reverse complements with low structural diversity are expressed together with the rRNA cluster. The three other hits are located between two coding genes in non-conserved genomic loci. Two of them are independently expressed from their surrounding genes and are structurally diverse. In summary, we found that Bacillaceae-1 RNA motifs upstream flanking of ribosomal RNA clusters tend to have one stable structure with the reverse complementary motif expressed in B. subtilis. In contrast, a subgroup of intergenic motifs has the thermodynamic potential for structural switches.
Abstract
Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of ...two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3′ UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.
In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such ...environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.
In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K
was elicited by high concentration of Na
in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.
The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.
The production of the alpha-amylase (AMY) enzyme in
Bacillus subtilis
at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA ...chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes
via
gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.
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