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Presents research reporting that the cyanobacterium `Calothrix' encodes sulphur-depleted versions of its most abundant proteins, phycocyanin and its auxiliary polypeptides, which it specifically expresses under conditions of sulphur limitation. Provides direct evidence that the structure of macromolecules can be subject to metabolic optimization. Research methods.

The cyanobacterium Calothrix PCC 7601 thrB gene, encoding homoserine kinase (EC 2.7.1.39), was cloned via complementation of an Escherichia coli threonine auxotroph, and its nucleotide sequence was determined. The comparison of the homoserine kinase amino acid sequences from Calothrix PCC 7601, E. coli K12 and Bacillus subtilis 168 indicates a closer relationship between cyanobacteria and bacillaceae than between cyanobacteria and enterobacteriaceae. Sequence analysis of the 5′ and 3′ flanking regions of the Calothrix thrB gene revealed the existence of a 169-codon-long open reading frame downstream from thrB: this sequence may be the second gene of a Calothrix thr operon. Two types of tandemly repeated sequences, sharing similarities with other prokaryotic transcriptional regulatory elements, were detected in the region upstream from the thrB gene. [ABSTRACT FROM AUTHOR]

Abstract Background Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. Results Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. Conclusions Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of ?-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of ?-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. [ABSTRACT FROM AUTHOR]

Multiple antibiotic resistances are a major global health threat. The predominant tool for adaptation in Gram-negative bacteria is the integron. Under stress, it rearranges gene cassettes to offer an escape using the tyrosine recombinase IntI, recognizing folded DNA hairpins, the attC sites. Four recombinases and two attC sites form the synaptic complex. Yet, for unclear reasons, the recombination efficiency varies greatly. Here, we established an optical tweezers force spectroscopy assay to probe the synaptic complex stability and revealed, for seven combinations of attC sites, significant variability in the mechanical stability. We found a strong correlation between mechanical stability and recombination efficiency of attC sites in vivo, indicating a regulatory mechanism from the DNA structure to the macromolecular complex stability. Taking into account known forces during DNA metabolism, we propose that the variation of the integron in vivo recombination efficiency is mediated by the synaptic complex stability. We anticipate that further recombination processes are also affected by their corresponding mechanical stability.

Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). The coordinated segregation of the replication terminus of both chromosomes (TerI and TerII) determines when and where cell division occurs. ChrI encodes a homologue of Escherichia coli MatP, a protein that binds to a DNA motif (matS) that is overrepresented in replication termini. Here, we use a combination of deep sequencing and fluorescence microscopy techniques to show that V. cholerae MatP structures TerI and TerII into macrodomains, targets them to mid-cell during replication, and delays their segregation, thus supporting that ChrII behaves as a bona fide chromosome. We further show that the extent of the segregation delay mediated by MatP depends on the number and local density of matS sites, and is independent of its assembly into tetramers and any interaction with the divisome, in contrast to what has been previously observed in E. coli., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Integrons are genetic elements that increase the evolvability of bacteria by capturing new genes and stockpiling them in arrays. Sedentary chromosomal integrons (SCIs) can be massive and highly stabilized structures encoding hundreds of genes, whose function remains generally unknown. SCIs have co-evolved with the host for aeons and are highly intertwined with their physiology from a mechanistic point of view. But, paradoxically, other aspects, like their variable content and location within the genome, suggest a high genetic and functional independence. In this work, we have explored the connection of SCIs to their host genome using as a model the Superintegron (SI), a 179-cassette long SCI in the genome of Vibrio cholerae N16961. We have relocated and deleted the SI using SeqDelTA, a novel method that allows to counteract the strong stabilization conferred by toxin-antitoxin systems within the array. We have characterized in depth the impact in V. cholerae's physiology, measuring fitness, chromosome replication dynamics, persistence, transcriptomics, phenomics, natural competence, virulence and resistance against protist grazing. The deletion of the SI did not produce detectable effects in any condition, proving that-despite millions of years of co-evolution-SCIs are genetically and functionally isolated units of genomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

From April 20 to 23, 2024, three hundred ten researchers from around the world gathered in Saint-Malo, France, at the fourth International Congress on Transposable Elements (ICTE 2024), to present their most recent discoveries on transposable elements (TEs) and exchange ideas and methodologies. ICTE has been held every four years since 2008 (except in 2020, when it was exceptionally transformed into a seminar series due to the Covid-19 pandemic) and is organized by the French network on Mobile Genetic Elements (CNRS GDR 3546). This fourth edition offered two keynote presentations and four sessions presenting the latest findings and encouraging discussions on the following topics: (1) TEs, genome evolution and adaptation; (2) TEs in health and diseases; (3) TE control and epigenetics; (4) Transposition mechanisms and applications. The 2024 edition also included a half-day satellite workshop on new challenges in TE annotation, organized in collaboration with the TE Hub. The meeting gathered long-term TE enthusiasts, as well as newcomers to the field, with 77% of the participants attending ICTE for the first time., (© 2024. The Author(s).)

Background: Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium., Results: In this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens., Conclusion: We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae., (© 2024. The Author(s).)

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach., (© 2023 John Wiley & Sons Ltd.)

The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, linking the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics remains challenging due to the trade-offs between the resolution and the field of view. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Le Quellec et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Richard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 10 5 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

SUMMARYAminoglycosides (AGs) are long-known molecules successfully used against Gram-negative pathogens. While their use declined with the discovery of new antibiotics, they are now classified as critically important molecules because of their effectiveness against multidrug-resistant bacteria. While they can efficiently cross the Gram-negative envelope, the mechanism of AG entry is still incompletely understood, although this comprehension is essential for the development of new therapies in the face of the alarming increase in antibiotic resistance. Increasing antibiotic uptake in bacteria is one strategy to enhance effective treatments. This review aims, first, to consolidate old and recent knowledge about AG uptake; second, to explore the connection between AG-dependent bacterial stress and drug uptake; and finally, to present new strategies of potentiation of AG uptake for more efficient antibiotic therapies. In particular, we emphasize on the connection between sugar transport and AG potentiation., Competing Interests: The authors declare no conflict of interest.

Importance: The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides (AGs) in anaerobic conditions, but the mechanism is unknown. AGs are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the ravA-viaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in Vibrio cholerae . We show that membrane stress response systems Cpx and Zra2 are required in the absence of RavA-ViaA, for the tolerance to AGs and for outer membrane integrity. In the absence of these systems, the ∆ravvia strain's membrane becomes permeable to external agents such as the antibiotic vancomycin., Competing Interests: The authors declare no conflict of interest.

To preserve the integrity of their genome, bacteria rely on several genome maintenance mechanisms that are co-ordinated with the cell cycle. All members of the Vibrio family have a bipartite genome consisting of a primary chromosome (Chr1) homologous to the single chromosome of other bacteria such as Escherichia coli and a secondary chromosome (Chr2) acquired by a common ancestor as a plasmid. In this review, we present our current understanding of genome maintenance in Vibrio cholerae , which is the best-studied model for bacteria with multi-partite genomes. After a brief overview on the diversity of Vibrio genomic architecture, we describe the specific, common, and co-ordinated mechanisms that control the replication and segregation of the two chromosomes of V. cholerae . Particular attention is given to the unique checkpoint mechanism that synchronizes Chr1 and Chr2 replication., Competing Interests: The authors declare no conflict of interest.

Plasmids facilitate the vertical and horizontal spread of antimicrobial resistance genes between bacteria. The host range and adaptation of plasmids to new hosts determine their impact on the spread of resistance. In this work, we explore the mechanisms driving plasmid adaptation to novel hosts in experimental evolution. Using the small multicopy plasmid pB1000, usually found in Pasteurellaceae , we studied its adaptation to a host from a different bacterial family, Escherichia coli. We observed two different mechanisms of adaptation. One mechanism is single nucleotide polymorphisms (SNPs) in the origin of replication ( oriV ) of the plasmid, which increase the copy number in E. coli cells, elevating the stability, and resistance profile. The second mechanism consists of two insertion sequences (ISs), IS 1 and IS 10 , which decrease the fitness cost of the plasmid by disrupting an uncharacterized gene on pB1000 that is harmful to E. coli. Both mechanisms increase the stability of pB1000 independently, but only their combination allows long-term maintenance. Crucially, we show that the mechanisms have a different impact on the host range of the plasmid. SNPs in oriV prevent the replication in the original host, resulting in a shift of the host range. In contrast, the introduction of ISs either shifts or expands the host range, depending on the IS. While IS 1 leads to expansion, IS 10 cannot be reintroduced into the original host. This study gives new insights into the relevance of ISs in plasmid-host adaptation to understand the success in spreading resistance. IMPORTANCE ColE1-like plasmids are small, mobilizable plasmids that can be found across at least four orders of Gammaproteobacteria and are strongly associated with antimicrobial resistance genes. Plasmid pB1000 carries the gene bla ROB-1 , conferring high-level resistance to penicillins and cefaclor. pB1000 has been described in various species of the family Pasteurellaceae , for example, in Haemophilus influenzae, which can cause diseases such as otitis media, meningitis, and pneumonia. To understand the resistance spread through horizontal transfer, it is essential to study the mechanisms of plasmid adaptation to novel hosts. In this work we identify that a gene from pB1000, which encodes a peptide that is toxic for E. coli, and the low plasmid copy number (PCN) of pB1000 in E. coli cells are essential targets in the described plasmid-host adaptation and therefore limit the spread of pB1000-encoded bla ROB-1 . Furthermore, we show how the interplay of two adaptation mechanisms leads to successful plasmid maintenance in a different bacterial family., Competing Interests: The authors declare no conflict of interest.

It is unclear how gene order within the chromosome influences genome evolution. Bacteria cluster transcription and translation genes close to the replication origin ( oriC ). In Vibrio cholerae, relocation of s10-spc-α locus (S10), the major locus of ribosomal protein genes, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction in growth rate, fitness, and infectivity. To test the long-term impact of this trait, we evolved 12 populations of V. cholerae strains bearing S10 at an oriC -proximal or an oriC -distal location for 1,000 generations. During the first 250 generations, positive selection was the main force driving mutation. After 1,000 generations, we observed more nonadaptative mutations and hypermutator genotypes. Populations fixed inactivating mutations at many genes linked to virulence: flagellum, chemotaxis, biofilm, and quorum sensing. Throughout the experiment, all populations increased their growth rates. However, those bearing S10 close to oriC remained the fittest, indicating that suppressor mutations cannot compensate for the genomic position of the main ribosomal protein locus. Selection and sequencing of the fastest-growing clones allowed us to characterize mutations inactivating, among other sites, flagellum master regulators. Reintroduction of these mutations into the wild-type context led to a ≈10% growth improvement. In conclusion, the genomic location of ribosomal protein genes conditions the evolutionary trajectory of V. cholerae. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and evolution. A lack of suppression enables artificial gene relocation as a tool for genetic circuit reprogramming. IMPORTANCE The bacterial chromosome harbors several entangled processes such as replication, transcription, DNA repair, and segregation. Replication begins bidirectionally at the replication origin ( oriC ) until the terminal region ( ter ) organizing the genome along the ori-ter axis gene order along this axis could link genome structure to cell physiology. Fast-growing bacteria cluster translation genes near oriC . In Vibrio cholerae, moving them away was feasible but at the cost of losing fitness and infectivity. Here, we evolved strains harboring ribosomal genes close or far from oriC . Growth rate differences persisted after 1,000 generations. No mutation was able to compensate for the growth defect, showing that ribosomal gene location conditions their evolutionary trajectory. Despite the high plasticity of bacterial genomes, evolution has sculpted gene order to optimize the ecological strategy of the microorganism. We observed growth rate improvement throughout the evolution experiment that occurred at expense of energetically costly processes such the flagellum biosynthesis and virulence-related functions. From the biotechnological point of view, manipulation of gene order enables altering bacterial growth with no escape events.

Candida albicans is a major fungal pathogen of humans. Although its genome has been sequenced more than two decades ago, there are still over 4300 uncharacterized C. albicans genes. We previously generated an ORFeome as well as a collection of destination vectors to facilitate overexpression of C. albicans ORFs. Here, we report the construction of ∼2500 overexpression mutants and their evaluation by in vitro spotting on rich medium and in a liquid pool experiment in rich medium, allowing the identification of genes whose overexpression has a fitness cost. The candidates were further validated at the individual strain level. This new resource allows large-scale screens in different growth conditions to be performed routinely. Altogether, based on the concept of identifying functionally related genes by cluster analysis, the availability of this overexpression mutant collection will facilitate the characterization of gene functions in C. albicans., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Inteins are proteins embedded into host proteins from which they are excised in an autocatalytic reaction. Specifically, split inteins are separated into two independent fragments that reconstitute the host protein during the catalytic process. We recently developed a novel strategy for the specific killing of pathogenic and antibiotic resistant bacteria based on toxin-intein combinations. Bacterial type II toxin-antitoxin systems are protein modules in which the toxin can provoke cell death whereas the antitoxin inhibits toxin activity. Although our previous system was based on a split intein (iDnaE) and the CcdB toxin, we demonstrated that iDnaE is able to reconstitute four different toxins. To expand the applicability of our system by widening the repertoire of toxin-intein combinations for complex set-ups, we introduced a second intein, iDnaX, which was artificially split. We demonstrate that iDnaX is able to reconstitute the four toxins, and we manage to reduce its scar size to facilitate their use. In addition, we prove the orthogonality of both inteins (iDnaE and iDnaX) through a toxin reconstitution assay, thus opening the possibility for complex set-ups based on these toxin-intein modules. This could be used to develop specific antimicrobial and other biotechnological applications.