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1.
World J Microbiol Biotechnol ; 40(10): 319, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39261358

RESUMO

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.


Assuntos
Cromossomos Bacterianos , Elementos de DNA Transponíveis , Nisina , Streptococcus pneumoniae , Nisina/farmacologia , Nisina/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/efeitos dos fármacos , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , Regulação Bacteriana da Expressão Gênica , Enterococcus/genética , Enterococcus/efeitos dos fármacos , Vetores Genéticos/genética , Conjugação Genética , Streptococcus/genética , Streptococcus/efeitos dos fármacos , Streptococcus/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
2.
BMC Infect Dis ; 24(1): 942, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251928

RESUMO

BACKGROUND: Bacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis. METHODS: The complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes. RESULT: The distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361. CONCLUSION: The study identified thirty chromosome-encoded genes specific to B. anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.


Assuntos
Bacillus anthracis , Cromossomos Bacterianos , Genoma Bacteriano , Reação em Cadeia da Polimerase Multiplex , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Cromossomos Bacterianos/genética , Marcadores Genéticos , Antraz/microbiologia , Antraz/diagnóstico , Humanos , Sequenciamento Completo do Genoma/métodos
3.
Curr Microbiol ; 81(10): 305, 2024 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-39133322

RESUMO

The bacterium Bacillus subtilis is a widely used study model and industrial workhorse organism that belongs to the group of gram-positive bacteria. In this study, we report the analysis of a newly sequenced complete genome of B. subtilis strain SRCM117797 along with a comparative genomics of a large collection of B. subtilis strain genomes. B. subtilis strain SRCM117797 has 4,255,638 bp long chromosome with 43.4% GC content and high coding sequence association with macromolecules, metabolism, and phage genes. Genomic diversity analysis of 232 B. subtilis strains resulted in the identification of eight clusters and three singletons. Of 147 B. subtilis strains included, 89.12% had strain-specific genes, of which 6.75% encoded strain-specific insertion sequence family transposases. Our analysis showed a potential role of strain-specific insertion sequence family transposases in intra-cellular accumulation of strain-specific genes. Furthermore, the chromosomal layout of the core genes was biased: overrepresented on the upper half (closer to the origin of replication) of the chromosome, which may explain the fast-growing characteristics of B. subtilis. Overall, the study provides a complete genome sequence of B. subtilis strain SRCM117797, show an extensive genomic diversity of B. subtilis strains and insights into strain diversification mechanism and non-random chromosomal layout of core genes.


Assuntos
Bacillus subtilis , Genoma Bacteriano , Bacillus subtilis/genética , Filogenia , Variação Genética , Composição de Bases , Genômica , Cromossomos Bacterianos/genética , Análise de Sequência de DNA
4.
BMC Genomics ; 25(1): 774, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39118048

RESUMO

BACKGROUND: Pseudomonas juntendi is a newly identified opportunistic pathogen, of which we have limited understanding. P. juntendi strains are often multidrug resistant, which complicates clinical management of infection. METHODS: A strain of Pseudomonas juntendi (strain L4326) isolated from feces was characterized by MALDI-TOF-MS and Average Nucleotide Identity BLAST. This strain was further subject to whole-genome sequencing and Maximum Likelihood phylogenetic analysis. The strain was phenotypically characterized by antimicrobial susceptibility testing and conjugation assays. RESULTS: We have isolated the novel P. juntendi strain L4236, which was multidrug resistant, but retained sensitivity to amikacin. L4236 harbored a megaplasmid that encoded blaOXA-1 and a novel blaIMP-1 resistance gene variant. P. juntendi strain L4236 was phylogenetically related to P. juntendi strain SAMN30525517. CONCLUSION: A rare P. juntendi strain was isolated from human feces in southern China with a megaplasmid coharboring blaIMP-1-like and blaOXA-1. Antimicrobial selection pressures may have driven acquisition of drug-resistance gene mutations and carriage of the megaplasmid.


Assuntos
Farmacorresistência Bacteriana Múltipla , Filogenia , Plasmídeos , Pseudomonas , beta-Lactamases , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Plasmídeos/genética , beta-Lactamases/genética , Farmacorresistência Bacteriana Múltipla/genética , China , Humanos , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Sequenciamento Completo do Genoma , Fezes/microbiologia , Cromossomos Bacterianos/genética , Genoma Bacteriano
5.
Sci Rep ; 14(1): 19026, 2024 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-39152186

RESUMO

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.


Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Replicação do DNA , Proteínas de Ligação a DNA , Complexos Multiproteicos , Mycobacterium smegmatis , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Adenosina Trifosfatases/metabolismo , Complexos Multiproteicos/metabolismo , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
6.
Microbiol Spectr ; 12(9): e0050124, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39101706

RESUMO

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure "ISAba1-blaADC-25." The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.IMPORTANCEThe blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.


Assuntos
Acinetobacter baumannii , Antibacterianos , Carbapenêmicos , Farmacorresistência Bacteriana Múltipla , beta-Lactamases , Humanos , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/tratamento farmacológico , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , beta-Lactamases/genética , Carbapenêmicos/farmacologia , Cromossomos Bacterianos/genética , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Sequenciamento Completo do Genoma
7.
Antimicrob Agents Chemother ; 68(9): e0081724, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39133024

RESUMO

Escherichia coli ST131 is a multidrug-resistant lineage associated with the global spread of extended-spectrum ß-lactamase-producing organisms. Particularly, ST131 clade C1 is the most predominant clade in Japan, harboring blaCTX-M-14 at a high frequency. However, the process of resistance gene acquisition and spread remains unclear. Here, we performed whole-genome sequencing of 19 E. coli strains belonging to 12 STs and 12 fimH types collected between 1997 and 2016. Additionally, we analyzed the full-length genome sequences of 96 ST131-H30 clade C0 and C1 strains, including those obtained from this study and those registered in public databases, to understand how ST131 clade C1 acquired and spread blaCTX-M-14. We detected conjugative IncFII plasmids and IncB/O/K/Z plasmids carrying blaCTX-M-14 in diverse genetic lineages of E. coli strains from the 1990s to the 2010s, suggesting that these plasmids played an important role in the spread of blaCTX-M-14. Molecular phylogenetic and molecular clock analyses of the 96 ST131-H30 clade C0 and C1 strains identified 8 subclades. Strains harboring blaCTX-M-14 were clustered in subclades 4 and 5, and it was inferred that clade C1 acquired blaCTX-M-14 around 1993. All 34 strains belonging to subclade 5 possessed blaCTX-M-14 with ISEcp1 upstream at the same chromosomal position, indicating their common ancestor acquired blaCTX-M-14 in a single ISEcp1-mediated transposition event during the early formation of the subclade around 1999. Therefore, both the horizontal transfer of plasmids carrying blaCTX-M-14 to diverse genetic lineages and chromosomal integration in the predominant genetic lineage have contributed to the spread of blaCTX-M-14.


Assuntos
Farmacorresistência Bacteriana Múltipla , Escherichia coli , beta-Lactamases , Humanos , Antibacterianos/farmacologia , beta-Lactamases/genética , Cromossomos Bacterianos/genética , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Japão , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/genética , Sequenciamento Completo do Genoma
8.
Methods Mol Biol ; 2851: 23-37, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39210169

RESUMO

Lactobacillus bulgaricus is an industrial strain that has been used in the dairy products since ancient times. Because of the difficulty of chromosomal gene manipulation, there have been few reports of gene deletion, insertion, or replacement. We have developed a system that enables chromosomal gene manipulation of L. bulgaricus using a conjugal transfer vector and easily vector construction in E. coli. As an example, we have deleted a regulatory gene for the extracellular polysaccharide synthesis of L. bulgaricus to elucidate the function of the gene in question. Methods for constructing vectors for chromosomal integration, conjugation experiment, and obtaining deletion strains by double recombination were presented in detail. This conjugative shuttle vector, pGMß1, has been deposited at Addgene ( https://www.addgene.org )and can be used by anyone for academic purposes.


Assuntos
Cromossomos Bacterianos , Vetores Genéticos , Lactobacillus delbrueckii , Lactobacillus delbrueckii/genética , Vetores Genéticos/genética , Cromossomos Bacterianos/genética , Conjugação Genética , Escherichia coli/genética , Plasmídeos/genética , Deleção de Genes , Engenharia Genética/métodos
9.
Eur J Clin Microbiol Infect Dis ; 43(9): 1855-1860, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38958811

RESUMO

Leminorella grimontii strain LG-KP-E1-2-T0 was isolated from Zophobas morio larvae. It showed a susceptibility phenotype compatible with the expression of an inducible extended-spectrum ß-lactamase. The presence of a chromosomal bla gene encoding for the class A GRI-1 ß-lactamase was revealed by whole-genome sequencing. GRI-1 shared the highest amino acid identity with RIC-1 and OXY-type ß-lactamases (76-80%). Analysis of six further publicly-available L. grimontii draft genomes deposited in NCBI revealed that blaGRI-1 was always present. Core-genome analysis indicated that LG-KP-E1-2-T0 was unique from other strains. We provided the first complete genome of L. grimontii and new insights on its chromosomal ß-lactamases.


Assuntos
Genoma Bacteriano , beta-Lactamases , beta-Lactamases/genética , Animais , Cromossomos Bacterianos/genética , Sequenciamento Completo do Genoma , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Genômica , Larva/microbiologia , Larva/genética , Mariposas/microbiologia
10.
J Microbiol Methods ; 224: 106999, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39033856

RESUMO

In this study, we describe a novel method for one-step cloning and targeted duplication of P. ananatis chromosomal fragments. According to this method, the chromosomal region of interest is subcloned in vivo via λ Red recombination into the short synthetic non-replicable DNA fragment containing the excisable antibiotic-resistance marker gene and φ80 att-P site. The resulting circular non-replicating DNA molecule was immediately inserted into an alternative chromosomal locus due to φ80-integrase activity. To this end, the specially designed helper plasmid pONI, which can provide both the λ Red recombineering and φ80-integrase-mediated insertion, was constructed. In the described method, PCR amplification of the cloning fragment is unnecessary, making it convenient for manipulation of long-length DNA. Additionally, the possibility of spontaneous mutations occurring is completely precluded. This method was effectively used for the targeted chromosomal integration of additional copies of individual genes and operons up to 16 kb in size.


Assuntos
Cromossomos Bacterianos , Clonagem Molecular , Pantoea , Plasmídeos , Pantoea/genética , Clonagem Molecular/métodos , Cromossomos Bacterianos/genética , Plasmídeos/genética , DNA Bacteriano/genética , Recombinação Genética , Integrases/genética
11.
J Glob Antimicrob Resist ; 38: 275-280, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38996871

RESUMO

OBJECTIVES: The mechanisms underlying chromosomally encoded colistin resistance in Escherichia coli remain insufficiently investigated. In this study, we investigated the contribution of various pmrB mutations from E. coli clinical isolates to colistin resistance. METHODS: The resistance mechanisms in eight mcr-negative colistin-resistant E. coli isolates obtained from a nationwide surveillance program in Taiwan using recombinant DNA techniques and complementary experiments were investigated. The minimal inhibitory concentrations (MICs) of colistin in the recombinant strains were compared with those in the parental strains. The expression levels of pmrA and pmrK (which are part of the pmrCAB and pmrHFIJKLM operons associated with colistin resistance) were measured using reverse transcription-quantitative real-time polymerase chain reaction. RESULTS: In the complementation experiments, various mutated pmrB alleles from the eight mcr-negative colistin-resistant E. coli strains were introduced into an ATCC25922 mutant with a PmrB deletion, which resulted in colistin resistance. The MIC levels of colistin in the most complemented strains were comparable to those of the parental colistin-resistant strains. Increased expression levels of pmrA and pmrK were consistently detected in most complemented strains. The impact for colistin resistance was confirmed for various novel amino acid substitutions, P94L, G19E, L194P, L98R and R27L in PmrB from the parental clinical strains. The detected amino acid substitutions are distributed in the different functional domains of PmrB. CONCLUSIONS: Colistin resistance mediated by amino acid substitutions in PmrB is a major chromosomally encoded mechanism in E. coli of clinical origin.


Assuntos
Antibacterianos , Colistina , Farmacorresistência Bacteriana , Escherichia coli , Testes de Sensibilidade Microbiana , Mutação , Colistina/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Humanos , Proteínas de Bactérias/genética , Infecções por Escherichia coli/microbiologia , Taiwan , Cromossomos Bacterianos/genética , Proteínas de Escherichia coli/genética , Teste de Complementação Genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição
12.
Nat Commun ; 15(1): 5526, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38951499

RESUMO

An international collection of Staphylococcus aureus of clonal complex (CC) 398 from diverse hosts spanning all continents and a 30 year-period is studied based on whole-genome sequencing (WGS) data. The collection consists of publicly available genomic data from 2994 strains and 134 recently sequenced Swiss methicillin-resistant S. aureus (MRSA) CC398 strains. A time-calibrated phylogeny reveals the presence of distinct phylogroups present in Asia, North and South America and Europe. European MRSA diverged from methicillin-susceptible S. aureus (MSSA) at the beginning of the 1950s. Two major European phylogroups (EP4 and EP5), which diverged approximately 1974, are the main drivers of MRSA CC398 spread in Europe. Within EP5, an emergent MRSA lineage spreading among the European horse population (EP5-Leq) diverged approximately 1996 from the pig lineage (EP5-Lpg), and also contains human-related strains. EP5-Leq is characterized by staphylococcal cassette chromosome mec (SCCmec) IVa and spa type t011 (CC398-IVa-t011), and EP5-Lpg by CC398-SCCmecVc-t011. The lineage-specific antibiotic resistance and virulence gene patterns are mostly mediated by the acquisition of mobile genetic elements like SCCmec, S. aureus Genomic Islands (SaGIs), prophages and transposons. Different combinations of virulence factors are present on S. aureus pathogenicity islands (SaPIs), and novel antimicrobial resistance gene containing elements are associated with certain lineages expanding in Europe. This WGS-based analysis reveals the actual evolutionary trajectory and epidemiological trend of the international MRSA CC398 population considering host, temporal, geographical and molecular factors. It provides a baseline for global WGS-based One-Health studies of adaptive evolution of MRSA CC398 as well as for local outbreak investigations.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Filogenia , Infecções Estafilocócicas , Sequenciamento Completo do Genoma , Animais , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/epidemiologia , Humanos , Europa (Continente)/epidemiologia , Cavalos/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/classificação , Staphylococcus aureus/patogenicidade , Genoma Bacteriano , Fatores de Virulência/genética , Cromossomos Bacterianos/genética , Evolução Molecular , Suínos
13.
Methods Mol Biol ; 2819: 3-26, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028499

RESUMO

The complex architecture of DNA within living organisms is essential for maintaining the genetic information that dictates their functions and characteristics. Among the many complexities of genetic material organization, the folding and arrangement of DNA into chromosomes play a critical role in regulating gene expression, replication, and other essential cellular processes. Bacteria, despite their apparently simple cellular structure, exhibit a remarkable level of chromosomal organization that influences their adaptability and survival in diverse environments. Understanding the three-dimensional arrangement of bacterial chromosomes has long been a challenge due to technical limitations, but the development of Chromosome Conformation Capture (3C) methods revolutionized our ability to explore the hierarchical structure and the dynamics of bacterial genomes. Here, we review the major advances in the field of bacterial chromosome structure using 3C technology over the past decade.


Assuntos
Cromossomos Bacterianos , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Conformação de Ácido Nucleico , Genoma Bacteriano , Bactérias/genética
14.
Methods Mol Biol ; 2819: 125-146, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028505

RESUMO

Many approaches for measuring three-dimensional chromosomal conformations rely upon formaldehyde crosslinking followed by subsequent proximity ligation, a family of methods exemplified by 3C, Hi-C, etc. Here we provide an alternative crosslinking-free procedure for high-throughput identification of long-range contacts in the chromosomes of enterobacteria, making use of contact-dependent transposition of phage Mu to identify distant loci in close contact. The procedure described here will suffice to provide a comprehensive map of transposition frequencies between tens of thousands of loci in a bacterial genome, with the resolution limited by the diversity of the insertion site library used and the sequencing depth applied.


Assuntos
Mapeamento Cromossômico , Cromossomos Bacterianos , Escherichia coli , Escherichia coli/genética , Cromossomos Bacterianos/genética , Mapeamento Cromossômico/métodos , Bacteriófago mu/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Elementos de DNA Transponíveis/genética
15.
Methods Mol Biol ; 2819: 157-187, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028507

RESUMO

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.


Assuntos
Escherichia coli , Biologia Sintética , Escherichia coli/genética , Biologia Sintética/métodos , Clonagem Molecular/métodos , Engenharia Genética/métodos , Replicon/genética , Cromossomos Bacterianos/genética , Plasmídeos/genética , Cromossomos/genética
16.
Methods Mol Biol ; 2819: 611-623, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028526

RESUMO

The chromosome of a bacterium consists of a mega-base pair-long circular DNA, which self-organizes within the micron-sized bacterial cell volume, compacting itself by three orders of magnitude. Unlike eukaryotic chromosomes, it lacks a nuclear membrane and freely floats in the cytosol confined by the cell membrane. It is believed that strong confinement, cross-linking by associated proteins, and molecular crowding all contribute to determine chromosome size and morphology. Modelling the chromosome simply as a circular polymer decorated with closed side loops in a cylindrical confining volume has been shown to already recapture some of the salient properties observed experimentally. Here we describe how a computer simulation can be set up to study structure and dynamics of bacterial chromosomes using this model.


Assuntos
Cromossomos Bacterianos , Simulação de Dinâmica Molecular , Cromossomos Bacterianos/genética , DNA Bacteriano/genética
17.
Methods Mol Biol ; 2819: 625-653, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39028527

RESUMO

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication of genetic material. In a recent study, we presented a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics. This approach was used to investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell cycle. To achieve cell-scale chromosome structures that are realistic, we modeled the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. Additionally, the polymer interacts with ribosomes distributed according to cryo-electron tomograms of Syn3A. The polymer model was further augmented by computational models of loop extrusion by structural maintenance of chromosomes (SMC) protein complexes and topoisomerase action, and the modeling and analysis of multi-fork replication states.


Assuntos
Cromossomos Bacterianos , Replicação do DNA , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Bactérias/genética
18.
Nat Commun ; 15(1): 6018, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-39019870

RESUMO

In Escherichia coli, it is debated whether the two replisomes move independently along the two chromosome arms during replication or if they remain spatially confined. Here, we use high-throughput fluorescence microscopy to simultaneously determine the location and short-time-scale (1 s) movement of the replisome and a chromosomal locus throughout the cell cycle. The assay is performed for several loci. We find that (i) the two replisomes are confined to a region of ~250 nm and ~120 nm along the cell's long and short axis, respectively, (ii) the chromosomal loci move to and through this region sequentially based on their distance from the origin of replication, and (iii) when a locus is being replicated, its short time-scale movement slows down. This behavior is the same at different growth rates. In conclusion, our data supports a model with DNA moving towards spatially confined replisomes at replication.


Assuntos
Cromossomos Bacterianos , Replicação do DNA , DNA Bacteriano , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Microscopia de Fluorescência , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Origem de Replicação , Ciclo Celular/genética , DNA Polimerase Dirigida por DNA , Complexos Multienzimáticos
19.
Front Cell Infect Microbiol ; 14: 1410921, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39015336

RESUMO

Objective: The emergence of clinical Klebsiella pneumoniae strains harboring acrAB-tolC genes in the chromosome, along with the presence of two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 genes on a plasmid, has presented a significant clinical challenge. Methods: In order to study the detailed genetic features of K. pneumoniae strain SC35, both the bacterial chromosome and plasmids were sequenced using Illumina and nanopore platforms. Furthermore, bioinformatics methods were employed to analyze the mobile genetic elements associated with antibiotic resistance genes. Results: K. pneumoniae strain SC35 was found to possess a class A beta-lactamase and demonstrated resistance to all tested antibiotics. This resistance was attributed to the presence of efflux pump genes, specifically acrAB-tolC, on the SC35 chromosome. Additionally, the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65, as well as bla TEM-1 with rmtB, which shared overlapping structures with mobile genetic elements as In413, Tn3, and TnAs3. Through plasmid transfer assays, it was determined that the SC35 plasmid p1 could be successfully transferred with an average conjugation frequency of 6.85 × 10-4. Conclusion: The structure of the SC35 plasmid p1 appears to have evolved in correlation with other plasmids such as pKPC2_130119, pDD01754-2, and F4_plasmid pA. The infectious strain SC35 exhibits no susceptibility to tested antibioticst, thus effective measures should be taken to prevent the spread and epidemic of this strain.


Assuntos
Antibacterianos , Cromossomos Bacterianos , Infecções por Klebsiella , Klebsiella pneumoniae , Plasmídeos , beta-Lactamases , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Plasmídeos/genética , beta-Lactamases/genética , Infecções por Klebsiella/microbiologia , Antibacterianos/farmacologia , Cromossomos Bacterianos/genética , Humanos , Testes de Sensibilidade Microbiana , Sequências Repetitivas Dispersas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
20.
J Infect Dis ; 230(1): 231-238, 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39052728

RESUMO

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.


Assuntos
Proteínas de Bactérias , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/epidemiologia , Recombinases/genética , Recombinases/metabolismo , Mineração de Dados , Humanos
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