scholarly journals The ΦBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4784 ◽  
Author(s):  
Marion Herisse ◽  
Jessica L. Porter ◽  
Romain Guerillot ◽  
Takehiro Tomita ◽  
Anders Goncalves Da Silva ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Bacterial Species ◽  
Bacterial Attachment ◽  
Single Strain ◽  
Molecular Microbiology ◽  
Attachment Sites ◽  
Phage Integrase ◽  
Foreign Dna ◽  
Stable Vectors ◽  
Target Effects

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages ΦC31 and ΦBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the ΦBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on ΦBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a ΦBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that ΦBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

scholarly journals Inactivation of the SauI Type I Restriction-Modification System Is Not Sufficient To Generate Staphylococcus aureus Strains Capable of Efficiently Accepting Foreign DNA

2009 ◽  
Vol 75 (10) ◽  
pp. 3034-3038 ◽  
Author(s):  
Helena Veiga ◽  
Mariana G. Pinho
Keyword(s):  
Staphylococcus Aureus ◽  
Genetic Manipulation ◽  
Type I ◽  
Modification System ◽  
Single Strain ◽  
Pathogenic Organism ◽  
Restriction Modification System ◽  
High Transformation ◽  
Foreign Dna ◽  
Restriction Modification

ABSTRACT Genetic manipulation of Staphylococcus aureus is limited by the availability of only a single strain, RN4220, that is capable of easily accepting foreign DNA. Inactivation of the hsdR gene of the SauI type I restriction-modification system was shown previously to be responsible for the high transformation efficiency of RN4220 (D. E. Waldron and J. A. Lindsay, J Bacteriol. 188:5578-5585, 2006). However, deletion of this gene in three different S. aureus strains was not sufficient to make them readily transformable, which would be remarkably useful for genetic studies of this pathogenic organism. These results indicate that another unknown factor(s) is required for the transformable phenotype in S. aureus.

scholarly journals Discovery of novel community-relevant small proteins in a simplified human intestinal microbiome

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Hannes Petruschke ◽  
Christian Schori ◽  
Sebastian Canzler ◽  
Sarah Riesbeck ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Intestinal Microbiota ◽  
De Novo ◽  
Bacterial Species ◽  
Intestinal Microbiome ◽  
Single Strain ◽  
Small Proteins ◽  
Human Intestinal Microbiota ◽  
Wide Range

Abstract Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract.

scholarly journals Slow Genetic Divergence of Helicobacter pylori Strains during Long-Term Colonization

2005 ◽  
Vol 73 (8) ◽  
pp. 4818-4822 ◽  
Author(s):  
Annelie Lundin ◽  
Britta Björkholm ◽  
Ilya Kupershmidt ◽  
Magnus Unemo ◽  
Peter Nilsson ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Genetic Divergence ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Genetic Change ◽  
Genetic Changes ◽  
Single Strain ◽  
Divergent Gene ◽  
Asymptomatic Adult ◽  
H Pylori

ABSTRACT The genetic variability of Helicobacter pylori is known to be high compared to that of many other bacterial species. H. pylori is adapted to the human stomach, where it persists for decades, and adaptation to each host results in every individual harboring a distinctive bacterial population. Although clonal variants may exist within such a population, all isolates are generally genetically related and thus derived from a common ancestor. We sought to determine the rate of genetic change of H. pylori over 9 years in two asymptomatic adult patients. Arbitrary primed PCR confirmed the relatedness of individual subclones within a patient. Furthermore, sequencing of 10 loci (∼6,000 bp) in three subclones per time and patient revealed only two base pair changes among the subclones from patient I. All sequences were identical among the patient II subclones. However, PCR amplification of the highly divergent gene amiA revealed great variation in the size of the gene between the subclones within each patient. Thus, both patients harbored a single strain with clonal variants at both times. We also studied genetic changes in culture- and mouse-passaged strains, and under both conditions no genetic divergence was found. These results suggest that previous estimates of the rate of genetic change in H. pylori within an individual might be overestimates.

scholarly journals Salmonellaexpresses foreign genes during infection by degrading their silencer

2020 ◽  
Vol 117 (14) ◽  
pp. 8074-8082 ◽  
Author(s):  
Jeongjoon Choi ◽  
Eduardo A. Groisman
Keyword(s):  
Binding Sites ◽  
Bacterial Species ◽  
Foreign Gene ◽  
Regulatory Sequences ◽  
Intracellular Pathogen ◽  
Heat Stable ◽  
Serovar Typhimurium ◽  
Foreign Dna ◽  
Foreign Genes ◽  
Virulence Regulator

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogenSalmonella entericaserovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes—even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Relationship between the Physicochemical Properties of Nonchitinolytic Listeria and Salmonella and Their Attachment to Shrimp Carapace

2009 ◽  
Vol 72 (6) ◽  
pp. 1181-1189 ◽  
Author(s):  
M. N. WAN NORHANA ◽  
REBECCA M. GOULTER ◽  
SUSAN E. POOLE ◽  
HILTON C. DEETH ◽  
GARY A. DYKES
Keyword(s):  
Cell Surface ◽  
Physicochemical Properties ◽  
Surface Charge ◽  
Foodborne Pathogens ◽  
Bacterial Species ◽  
Chitinase Activity ◽  
Bacterial Attachment ◽  
Bacterial Cells ◽  
Production Chain ◽  
Cell Surface Charge

Listeria and Salmonella are important foodborne pathogens normally associated with the shrimp production chain. This study investigated the potential of Salmonella Typhimurium, Salmonella Senftenberg, and Listeria monocytogenes (Scott A and V7) to attach to and colonize shrimp carapace. Attachment and colonization of Listeria and Salmonella were demonstrated. Shrimp abdominal carapaces showed higher levels of bacterial attachment (P < 0.05) than did head carapaces. Listeria consistently exhibited greater attachment (P < 0.05) than did Salmonella on all surfaces. Chitinase activity of all strains was tested and found not to occur at the three temperatures (10, 25, and 37°C) tested. The surface physicochemical properties of bacterial cells and shrimp carapace were studied to determine their role in attachment and colonization. Salmonella had significantly (P < 0.05) more positive (−3.9 and −6.0 mV) cell surface charge than Listeria (−18 and −22.8 mV) had. Both bacterial species were found to be hydrophilic (<35%) when measured by the bacterial adherence to hydrocarbon method and by contact angle (θ) measurements (Listeria, 21.3 and 24.8°, and Salmonella, 14.5 and 18.9°). The percentage of cells retained by Phenyl-Sepharose was lower for Salmonella (12.8 to 14.8%) than it was for Listeria (26.5 to 31.4%). The shrimp carapace was found to be hydrophobic (θ = 74.5°), and a significant (P < 0.05) difference in surface roughness between carapace types was noted. There was a linear correlation between bacterial cell surface charge (r2 = 0.95) and hydrophobicity (r2 = 0.85) and initial attachment (P < 0.05) of Listeria and Salmonella to carapaces. However, the same properties could not be related to subsequent colonization.

scholarly journals Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli

2012 ◽  
Vol 445 (3) ◽  
pp. 383-392 ◽  
Author(s):  
Diana Munera ◽  
Eric Martinez ◽  
Svetlana Varyukhina ◽  
Arvind Mahajan ◽  
Jesus Ayala-Sanmartin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Host Cell Proteins ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Attachment Sites ◽  
Infected Cells

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na+/H+ exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

scholarly journals Avoiding CRISPR: Plasmid design for genetic engineering

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Genetic Engineering ◽  
Bacterial Species ◽  
Plasmid Vector ◽  
Defense System ◽  
Dna And Rna ◽  
Natural Defense ◽  
Plasmid Transduction ◽  
Protospacer Adjacent Motif ◽  
Foreign Dna ◽  
Foreign Genes

Clustered regularly interspersed short palindromic repeats (CRISPR) is a natural defense system for bacteria and archaea against foreign DNA and RNA. Specifically, short snippets of foreign DNA or RNA are incorporated into protospacer adjacent motif (PAM) repeats sequences in the genome of the bacterial species, and serve as molecular memory of past infections by viruses. These repeats are transcribed by RNA polymerases and perform constant surveillance of the bacterial cell cytoplasm for foreign DNA. Once detected, PAM sequences would bind to the foreign DNA leading to the recruitment of Cas endonuclease protein that cut the foreign DNA. Plasmids are double stranded DNA vectors that serve to carry foreign genes into the cell for genetic engineering. Hence, plasmids are also foreign DNA with respect to the CRISPR system of the cell. To avoid destruction by the Cas protein, plasmid should not contain sequences that would bind to any of the PAM sequences encoded in the genome of the bacterial species. Thus, the PAM sequences of each bacterial species where genetic engineering is to be performed should be sequenced, and the knowledge gained utilized in the design of plasmid vectors that do not carry any of the sequences encoded by PAM repeats. Such an approach would help reduce the chances of destruction of plasmid vector once it was introduced to the cell, and would help improve the efficiency of plasmid transduction and genetic engineering. Collectively, CRISPR is a natural cellular defense system that could destroy introduced plasmid vector through recognition by PAM repeat sequences encoded in the cell’s genome. Sequencing of the PAM sequence of the bacterial species followed by careful design of the plasmid DNA sequence would significantly reduce the chances of destruction of the vector by CRISPR once it was introduced into the cell.

scholarly journals Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

2017 ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits ◽  
Derivatives Of

AbstractIn biotechnological work horses likeStreptococcus thermophilusandBacillus subtilisnatural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starterLactococcus lactisnatural competence has not been established to date. However,in silicoanalysis of complete genome sequences of 43L. lactisstrains revealed complete late-competence gene-sets in 2L. lactissubspeciescremorisstrains (KW2 and KW10) and 8L. lactissubspecieslactisstrains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulatorcomXinL. lactissubsp.lactisKF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast,comXexpression inL. lactisNZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of thecomEA-ECoperon in strain KF147, abolished thecomXdriven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-induciblecomXexpression intonisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to otherL. lactisstrains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the speciesLactococcus lactisare very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

scholarly journals A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

2019 ◽  
Vol 10 (2) ◽  
pp. 605-611
Author(s):  
Fang Tao ◽  
Kai Zhao ◽  
Qianqian Zhao ◽  
Fangzhi Xiang ◽  
Guomin Han
Keyword(s):  
Aspergillus Flavus ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Fungal Growth ◽  
Egfp Expression ◽  
Regulation Mechanism ◽  
Site Specific ◽  
Site Specific Integration ◽  
Foreign Dna ◽  
Novel Strategy

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus. To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

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