Olivares J et al. (2012) Environ Microbiol "Overproduction of the multidrug efflux pump MexEF-OprN does not impair Pseudomonas aeruginosa fitness in competition tests, but produces specific changes in bacterial regulatory networks."

Alvarez-Ortega C, Rojo F, Martinez JL, Linares JF, Olivares J, Kohler T

[Environ Microbiol, 2012]

It is generally assumed that acquisition of antibiotic resistance leads to non-specific fitness costs. We have tested the alternative hypothesis that acquisition of antibiotic resistance may not always produce a general burden to the microorganisms, as measured in competition tests, but rather lead to specific changes in bacterial physiology. To this end we studied the effect of overproducing the multidrug efflux pump MexEF-OprN on Pseudomonas aeruginosa due to a constitutive activation of MexT, the transcriptional activator of the mexEF-oprN genes. We found that overexpression of MexEF-OprN does not cause a significant decrease in P.aeruginosa fitness in classical competition tests, indicating the absence of a large metabolic burden and that any possible negative effects might be observed only under specific conditions. Transcriptomic analyses revealed that overexpression of MexEF-OprN results in reduced expression of several quorum-sensing regulated genes. We traced back this phenotype to a delay in PQS production due to extrusion of kynurenine, a PQS precursor, through the efflux pump. Type VI secretion was also impaired. A Caenorhabditis elegans model demonstrated that overproduction of MexEF-OprN impairs virulence in P.aeruginosa. This effect was mainly due to the activity of the efflux pump, and not to MexT, despite the fact that the latter regulates Type III and Type VI secretion. Altogether, these data indicate that antibiotic resistance can produce modifications in the bacterial regulatory networks with relevant consequences for the bacterial behaviour in specific ecosystems, including the infected host.

Sanz-Garcia F et al. (2019) Antimicrob Agents Chemother "Analysis of the Pseudomonas aeruginosa aminoglycosides differential resistome allows defining genes simultaneously involved in intrinsic antibiotic resistance and virulence."

Hernando-Amado S, Olivares-Pacheco J, Sanz-Garcia F, Martinez JL, Blanco P, Alvarez-Ortega C

[Antimicrob Agents Chemother, 2019]

High-throughput screening of transposon insertion libraries is a useful strategy for unveiling bacterial genes whose inactivation results in an altered susceptibility to antibiotics. A potential drawback of these studies is they are usually based on just one model antibiotic for each structural family, under the assumption that the results can be extrapolated to all members of said family. To determine if this simplification is appropriate, we have analysed the susceptibility of mutants of <i>P. aeruginosa</i> to four aminoglycosides. Our results indicate that each mutation produces different effects in the susceptibility to the tested aminoglycosides, with only two mutants showing similar changes in the susceptibility to all studied aminoglycosides. This indicates that the role of a particular gene in the resistome of a given antibiotic should not be generalized to other members of the same structural family. Five aminoglycosides hypersusceptible mutants inactivating <i>glnD, hflK, PA2798, PA3016</i> and <i>hpf</i> were chosen for further analysis, in order to elucidate if a lower aminoglycoside susceptibility correlates with cross-hypersusceptibility to other antibiotics and with impaired virulence. Our results indicate that <i>glnD</i> inactivation leads to an increased cross-susceptibility to different antibiotics. The mutant in this gene is strongly impaired in virulence traits such as pyocyanin production, biofilm formation, elastase activity, swarming motility and the ability to kill <i>C. elegans</i> Thus, GlnD might be an interesting target for developing antibiotic coadjunvants with anti-resistance and anti-virulence properties against <i>P. aeruginosa.</i>

Delich, Cassandra et al. (2020) MicroPubl Biol

Hebbar, Shilpa, Delich, Cassandra, Zinovyeva, Anna, Dillon, Annabelle, Haskell, Dustin, Winans, Noah

[MicroPubl Biol, 2020]

MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally repress gene expression (Gebert and MacRae, 2018). While many miRNA genes and their families have been analyzed for function (Miska et al. 2007, Alvarez-Saavedra and Horvitz 2010), there are microRNA genes for which loss of function alleles have not yet been generated. There are no available alleles for the C. elegans mir-49 gene. Using CRISPR-Cas9 genome editing, we generated two deletion alleles, zen99 and zen102, that disrupt the C. elegans mir-49 gene (Fig 1A). mir-49(zen99) and mir-49(zen102) delete 56 base pairs and 58 base pairs from the mir-49 locus, respectively (Fig 1B and Fig 1C). Each deletion nearly completely removes both strands generated by the mir-49 locus, mir-49-3p and mir-49-5p. Both mir-49 alleles are homozygous viable and appear to be superficially wild type. Careful phenotypic analysis will be important to characterize the effects of the two mir-49 deletions.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Antika TR et al. (2023) J Biol Chem "Sequence-specific targeting of C. elegans C-Ala to the D-loop of tRNAAla."

Horng JC, Hsu HL, Nazilah KR, Wang CC, Wang TL, Wang SC, Antika TR, Chuang TH, Chrestella DJ, Wang SW, Tseng YK, Pan HC

[J Biol Chem, 2023]

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala_c) robustly binds both ligands. How Ce-C-Ala_c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala_c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala_c specifically recognized the conserved invariant base G¹⁸ in the D-loop of tRNA^Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala (Ce-C-Ala_m) robustly bound both tRNA^Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA^Ala.

Allison L Abbott et al. (2002) East Coast Worm Meeting "IDENTIFICATION AND FUNCTIONAL ANALYSIS OF microRNA GENES IN C. elegans AND D. melanogaster."

Daniel Hopkins, Lorenzo Sempere, Rosalind C Lee, Allison L Abbott, Ann Lavanway, Nicholas S Sokol, Peter T Williams, David Jewell, Victor Ambros

[East Coast Worm Meeting, 2002]

lin-4 and let-7 are the founding members of a new family of regulatory RNAs, the microRNAs, many dozens of which have been identified in worms, flies and humans 1-3 . A total of 60 C. elegans microRNA genes have been published previously2,3 . In order to develop a comprehensive catalog of C. elegans miRNAs, we are continuing to identify new miRNA sequences from C. elegans cDNA libraries and from genomic sequence conservation between C. elegans and C. briggsae. So far we have identified 65 distinct new microRNA genes, bringing the total to at least 125 in worms. We confirmed the expression of all of these new microRNA genes using Northern blot analysis and are characterizing their patterns of expression during development. 47 of the new miRNA sequences occur in single copy in the C. elegans genome, while the remaining 18 sequences are found in multiple loci. Some microRNA genes are clustered, suggesting coordinate regulation by a shared promoter. Using RNA folding programs, we find that approximately 70% of these new miRNAs are predicted to be processed from stem-loop hairpin structures, like lin-4 and let-7. However, a significant portion of these miRNAs are not predicted to fold into a hairpin precursor. Interestingly, both hairpin and non-hairpin miRNAs exhibit Dicer-dependent production of the ~22nt mature miRNA. To aid in analyzing the whole family of microRNA genes, we have developed web interfaces to customized high-throughput Blast and mfold servers, and microViewer, a Java program tailored for microRNA annotation. In order to obtain mutants in microRNA genes, we are generating deletion mutations (for details, see abstract by Alvarez-Saavedra et al. ) and analyzing candidate genetic loci that map in close proximity to cloned microRNA genes. Lastly, expression profiles of microRNAs conserved between flies and worms are being compared. For example, mir-1, 2 and 87 are expressed throughout development in both flies and worms whereas mir-34 is enriched at later stages. These experiments should help elucidate the biological function of members of this new class of regulatory molecules.

Blumenthal TE et al. (1994) Worm Breeder's Gazette "C. elegans U2AF 65."

Zorio DAR, Lea K, Blumenthal TE

[Worm Breeder's Gazette, 1994]

C. elegans U2AF65

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Eric A Miska et al. (2005) International Worm Meeting "Functional analysis of the microRNA genes of C. elegans"

Victor Ambros, Nelson Lau, David Bartel, Ezequiel Alvarez-Saavedra, Andrew Hellman, Eric A Miska, Bob Horvitz, Allison Abbott

[International Worm Meeting, 2005]

The heterochronic genes lin-4 and let-7 encode small (21-22 nt) non-protein coding regulatory RNAs. Strains carrying a mutation in either of these genes are heterochronic, displaying retarded development with some cell lineages having an altered temporal pattern of cell division and differentiation. lin-4 and let-7 normally inhibit translation of target genes that when mutated lead to a phenotype opposite that of lin-4 and let-7 mutants: precocious development and early expression of certain paths of cell division and differentiation. More recently, molecular and bioinformatic approaches have identified many genes encoding small RNAs in C. elegans, Drosophila and mammals. All of these genes encode 21-25 nt RNAs derived from longer transcripts that contain partially double-stranded RNAs. These small RNAs, termed microRNAs (miRNAs), define a large, new class. To understand the biology of the C. elegans microRNA genes, we decided to combine the generation of loss-of-function mutants with GFP expression studies and target prediction using bioinformatics. We have generated deletion strains corresponding to 92 microRNAs, covering the majority of known microRNA genes. We will present an overview of the classes of mutant phenotypes we have observed. (for information about the mir-35 and the let-7 families of microRNAs, see also abstracts by Alvarez-Saavedra et al. and Abbott et al.). One focus will be the issue of redundancy within families of microRNA genes. This study represents the first comprehensive analysis of microRNA function.