FEMS Microbiology Letters

Jia, Miaomiao; Li, Ping; Yan, Yong; Liu, Xuejuan; Gao, Lei; Zhu, Guoying; Chen, Zhongwen

doi: 10.1093/femsle/fnaf009pmid: 39824655

Non-O1/non-O139 (NOVC) strains inhabit aquatic environments and sporadically induce human illnesses. This study involved the virulence and antimicrobial genetic characterization of 176 NOVC strains, comprising 25 from clinical samples and 151 from environmental sources, collected between 2021 and 2023. The antimicrobial susceptibility of the examined NOVC population was predominantly high, exhibiting only poor susceptibility to colistin, with 89.2% resistance. The examination of virulence genes revealed that the majority of strains were positive for glucose metabolism (als gene) (169/176, 96.0%). Through multilocus sequence typing, the 176 NOVC strains were categorised into 121 sequence types, 79 of which were novel. NOVC strains demonstrate significant genetic variability and frequently engage in recombination. This work offers genetic characterization of the pathogenicity and antimicrobial resistance of a NOVC community. Our findings offer insights that may aid in the development of preventative and treatment methods for this pathogen.

Toya, Yoshihiro; Imada, Tatsumi; Ishibashi, Mai; Kawamoto, Yuichi; Isshiki, Kinuka; Shibai, Atsushi; Furusawa, Chikara; Shimizu, Hiroshi

doi: 10.1093/femsle/fnaf013pmid: 39870377

Phosphoenolpyruvate carboxylase encoded by ppc catalyzes the anaplerotic reaction of oxaloacetate in the tricarboxylic acid (TCA) cycle in Escherichia coli. Deletion of ppc does not prevent the cells from replenishing oxaloacetate via the glyoxylate shunt, but the ppc-deletion strain almost did not grow on glucose. In the present study, we obtained evolved strains by deleting both ppc and mutS to increase the mutation rate and investigated the mechanisms for improving growth by analyzing the mutated genes. Genome resequencing revealed that the evolved strains have non-synonymous mutations in icd encoding isocitrate dehydrogenase (ICDH). The introduction of icd mutations rescued the growth defects caused by ppc deletion. ICDH activity was strongly reduced by the amino acid substitutions G205D or N232S. The evolved strains appeared to suppress the competitive pathway for increasing the glyoxylate shunt flux. In metabolic engineering, the deletion of iclR, which encodes a repressor of the aceBAK operon, has been used to activate the glyoxylate shunt. The growth rate of the ΔppcΔiclR strain slightly increased, but it was still much lower than that of the Δppc + icdG205D strains. This finding suggests that iclR deletion is not sufficient to enhance glyoxylate shunt flux and that inactivation of the competitive pathway by icd mutations is more effective.

Takita, Kazumi; Someya, Nobutaka; Morohoshi, Tomohiro

doi: 10.1093/femsle/fnae117pmid: 39762131

Burkholderia gladioli produces a yellow-pigmented toxin called toxoflavin, and causes disease on a variety of plants. Previous studies have suggested that the pathogenicity of B. gladioli is regulated by an N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing (QS) system. In this study, complete genome sequencing revealed that B. gladioli pv. gladioli MAFF 302385 possesses two types of AHL synthase and AHL receptor gene pairs: glaI1/glaR1 and glaI2/glaR2. Disruption of QS genes revealed that the glaI1/glaR1 QS system regulated swarming motility, biofilm formation, and colony formation via N-octanoyl-l-homoserine lactone. Although Escherichia coli harboring glaI2 produced N-(3-hydroxyoctanoyl)-l-homoserine lactone and N-(3-hydroxydecanoyl)-l-homoserine lactone, the expression of glaI2 was not confirmed in MAFF 302385 cells. We also found that toxoflavin production was regulated by the glaI1/glaR1 QS system in liquid medium, but not on agar medium. When pathogenicity tests were performed on gladiolus leaves, the wild-type and QS mutants showed a similar level of disease. Our results demonstrated that only the glaI1/glaR1-mediated QS system is active in MAFF 302385, but major virulence factors, especially toxoflavin, are not completely dependent on the QS system.

Jain, Diamond; Ajith, Tejavath; Verma, Jyoti; Chatterjee, Debasmita; Ghosh, Anindya S

doi: 10.1093/femsle/fnaf007pmid: 39799381

Verona-integron-metallo-β-lactamase (VIM-2) is one of the most widespread class B β-lactamase responsible for β-lactam resistance. Although active-site residues help in metal binding, the residues nearing the active-site possess functional importance. Here, to decipher the role of such residues in the activity and stability of VIM-2, the residues E146, D182, N210, S207, and D213 were selected through in-silico analyses and substituted with alanine using site-directed mutagenesis. The effects of substitution mutations were assessed by comparing the changes in β-lactam susceptibility pattern of Escherichia coli host cell expressing VIM-2 and its mutated proteins. VIM-2_N210A enhanced the susceptibility of the host by ∼4–8 folds against penicillins and cephalosporins, while the expression of VIM-2_D182A radically increased the susceptibility of host. However, expression of VIM-2_E146A reduced the susceptibility of host by 2-fold. Further, proteins were purified to homogeneity, and VIM_N210A and VIM_D182A displayed reduced thermal stability than VIM-2. Moreover, in vitro catalytic efficiencies of VIM-2_D182A were drastically reduced against all the β-lactams tested whereas the same were moderately reduced for VIM-2_N210A. Conversely, the catalytic efficiency was marginally altered for VIM_E146A. Overall, we infer that both N210A and D182A substitutions negatively affect the performance of VIM-2 by influencing substrate specificity and stability, respectively.

Schweitzer, Matthias; Friedrich, Andrea Marianne; Dennig, Alexander; Berg, Gabriele; Müller Bogotá, Christina Andrea

doi: 10.1093/femsle/fnaf004pmid: 39805715

Terminal olefins are important platform chemicals, drop-in compatible hydrocarbons and also play an important role as biocontrol agents of plant pathogens. Currently, 1-alkenes are derived from petroleum, although microbial biosynthetic routes are known. Jeotgalicoccus sp. ATCC 8456 produces 1-alkenes via the fatty acid decarboxylase OleTJE. UndA and UndB are recently identified non-heme iron oxidases converting medium-chain fatty acids into terminal alkenes. Our knowledge about the diversity and natural function of OleTJE, UndA, and UndB homologs is scarce. We applied a combined screening strategy—solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME GC–MS) and polymerase chain reaction (PCR)-based amplification—to survey an environmental strain collection for microbial 1-alkene producers and their corresponding enzymes. Our results reinforce the high level of conservation of UndA and UndB genes across the genus Pseudomonas. In vivo production of defined 1-alkenes (C9–C13; C15; C19) was directed by targeted feeding of fatty acids. Lauric acid feeding enabled 1-undecene production to a concentration of 3.05 mg l−1 in Jeotgalicoccus sp. ATCC 8456 and enhanced its production by 105% in Pseudomonas putida 1T1 (1.10 mg l−1). Besides, whole genome sequencing of Jeotgalicoccus sp. ATCC 8456 enabled reconstruction of the 1-alkene biosynthetic pathway. These results advance our understanding of microbial 1-alkene synthesis and the underlying genetic basis.

Kulichevskaya, Irina S; Ivanova, Anastasia A; Naumoff, Daniil G; Zlatogorskaya, Anna A; Kachmazov, Gennady S; Dedysh, Svetlana N

doi: 10.1093/femsle/fnaf006pmid: 39805719

Aerobic chemoorganotrophic planctomycetes of the genus Schlesneria colonize a wide spectrum of freshwater ecosystems. The only described species of this genus, S. paludicola, is represented by ellipsoid-shaped, moderately acidophilic bacteria isolated from acidic peat bogs. Here, we characterize a novel neutrophilic member of this genus from a mountain wetland, strain T3-172T. This isolate possesses unpigmented, non-motile, spherical cells, which grow on media containing some mono- and polysaccharides in the pH range of 6.0–7.5 and at temperatures between 15 and 37°C. Good growth is observed on xylan, one of the major plant cell wall biopolymers. The genome of strain T3-172T is 7.16 Mb in size and contains two unlinked sets of rRNA genes, 93 tRNA genes, and about 5500 potential protein-coding genes. Among the latter, the GH5 family protein is identified as the most reliable candidate for the cleavage of xylan. The 16S rRNA gene sequence of strain T3-172T displays 97.8% similarity to that of S. paludicola MPL7T, and the average nucleotide identity between the genomes of these planctomycetes is 71.6%. We, therefore, propose a novel species of the genus Schlesneria, S. sphaerica sp. nov., with strain T3-172T (=KCTC 102306T = VKM B-3856T) as the type strain.

Luo, Dan; Ma, Juanjuan; Xie, Weile; Wang, Zhe

doi: 10.1093/femsle/fnaf011pmid: 39890598

Understanding bacterial responses to antibiotics is essential for identifying resistance mechanisms and developing novel therapies. This study evaluated the resistance of Staphylococcus aureus (S. aureus) to fusidic acid (FD) in 100 patients with skin and soft tissue infections (SSTIs), revealing susceptibility to FD despite resistance to other antibiotics. Through adaptive laboratory evolution, we developed a highly FD-resistant strain, E10, and identified three gene mutations (fusA, BPENGOFF-00211, and rplF) using whole-genome sequencing. The fusA mutation was the primary contributor to resistance. Furthermore, the evolved fusA mutant strain (H457Y) displayed impaired coagulation function and reduced growth rates. We also analyzed the metabolomic profiles of ancestral ATCC 25923 and evolved E10 strains, both treated and untreated with FD, revealing that the fusA gene can independently induce metabolic reprogramming. These changes primarily impacted pathways involved in central carbon metabolism, nucleotide metabolism, and amino acid synthesis. This study highlights the complexity of FD resistance in S. aureus and offers insights into the metabolic pathways associated with antibiotic resistance.

Akbulut, Sumeyye; Dasdemir, Elanur; Ozkan, Hakan; Adiguzel, Ahmet

doi: 10.1093/femsle/fnaf002pmid: 39814574

In this study designed to isolate lactic acid bacteria (LAB) with bacteriocin production potential, white cheese samples were collected from different provinces of Turkey and isolation was carried out. A series of experiments were carried out for the main purpose and the actual bacteriocin producers were identified by detecting the genes encoding this bacteriocin. The experiments carried out in this direction were initially carried out with 20 isolates and as a result of various experiments, the number of isolates was reduced to 8 and the study was continued with 8 isolates. In order to determine that the eight isolates identified as a result of a phenotypic and biochemical characterization study were true bacteriocin-producing strains, their antibacterial activity was investigated and then the presence of bacteriocin genes was examined by specific polymerase chain reaction (PCR) using gene-specific primers. As a result, MS16 coded Lactiplantibacillus plantarum OR922652 was found to have strong antibacterial activity against Escherichia coli, Klebsiella pneumonia, Yersinia enterocolitica, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus; the isolate was susceptible to clinically important antibiotics (ciprofloxacin, gentamicin, penicillin G, ampicillin, chloramphenicol, and vancomycin) and resistant to erythromycin, had no hemolytic activity and possessed plnA and plnD genes encoding bacteriocin production. In conclusion, the MS16 coded L. plantarum isolate has emerged as a promising strain that can be used especially in the health field and in the food industry related to LAB.

Kyrkou, Ifigeneia; Bartell, Jennifer; Lechuga, Ana; Lood, Cédric; Marvig, Rasmus L; Lavigne, Rob; Molin, Søren; Krogh Johansen, Helle

doi: 10.1093/femsle/fnaf017pmid: 39890605

Pseudomonas aeruginosa has increasing clinical relevance and commonly occupies the cystic fibrosis (CF) airways. Its ability to colonize and persist in diverse niches is attributed to its large accessory genome, where prophages represent a common feature and may contribute to its fitness and persistence. We focused on the CF airways niche and used 197 longitudinal isolates from 12 patients persistently infected by P. aeruginosa. We computationally predicted intact prophages for each longitudinal group and scored their long-term persistence. We then confirmed prophage inducibility and mapped their location in the host chromosome with lysate sequencing. Using comparative genomics, we evaluated prophage genomic diversity, long-term persistence, and level of genomic maintenance. Our findings support previous findings that most P. aeruginosa genomes harbour prophages some of which can self-induce, and that a common CF-treating antibiotic, ciprofloxacin, can induce prophages. Induced prophage genomes displayed high diversity and even genomic novelty. Finally, all induced prophages persisted long-term with their genomes avoiding gene loss and degradation over 4 years of host replication in the stressful CF airways niche. This and our detection of phage genes, which contribute to host competitiveness and adaptation, lends support to our hypothesis that the vast majority of prophages detected as intact and inducible in this study facilitated their host fitness and persistence.

Rumyantseva, Natalia A; Shutov, Vladimir M; Belenkaia, Dina G; Alekseev, Aleksandr A; Morozova, Natalia E; Vedyaykin, Alexey D

doi: 10.1093/femsle/fnaf010pmid: 39890601

SMC (Structural Maintenance of Chromosomes) ATPase proteins are integral components of complexes bearing the same name, crucial for the spatial organization of DNA across diverse life forms, spanning bacteria, archaea, and eukaryotes. It is proposed that in bacteria, SMC complexes facilitate DNA compaction through loop extrusion and aid in the segregation of daughter nucleoids. In this paper, the properties of the SMC ATPase protein from Ureaplasma parvum were investigated by using a spectrum of methods, including conventional biochemical methods as well as advanced single-molecule techniques. Our findings reveal distinctive properties of this protein compared to its extensively studied homologue from Bacillus subtilis. Notably, our results suggest that U. parvum Smc ATPase facilitates DNA compaction even in the absence of ATP.