International Journal of Molecular Sciences

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15 pages, 7094 KiB

Open AccessArticle

Lessons from RatA: Why the Basics in Molecular Biology Are Still Crucial!

by Michel Fasnacht, Denise Schratt and Isabella Moll

Int. J. Mol. Sci. 2025, 26(7), 3100; https://doi.org/10.3390/ijms26073100 - 27 Mar 2025

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Abstract

Since the first bacterial genomes were sequenced and annotated over 25 years ago, sequencing technologies have rapidly advanced in both speed and cost efficiency. To date, over two million annotated bacterial genomes have been deposited in the National Center for Biotechnology Information (NCBI) [...] Read more.

Since the first bacterial genomes were sequenced and annotated over 25 years ago, sequencing technologies have rapidly advanced in both speed and cost efficiency. To date, over two million annotated bacterial genomes have been deposited in the National Center for Biotechnology Information (NCBI) database. Yet, there are many genes with unknown functions and, furthermore, conflicting results have been published for many investigated genes. One example is the ratA (or pasT) gene from Escherichia coli (E. coli) K-12 strains. Initially identified as a ribosome-targeting toxin, later studies described RatA as the bacterial homolog of the mitochondrial Coq10 protein and, therefore, beneficial for E. coli cells during aerobic growth. This study shows that these conflicting results originated from a mis-annotation of the start codon in the genomic sequence. Overexpression of the ratA gene as currently annotated leads to the synthesis of two RatA protein variants, a toxic and a non-toxic one. This study further identifies the endogenous ratA promoter and shows that only the shorter, non-toxic variant of RatA is synthesized during different growth phases specifically under aerobic conditions. Our findings thereby not only solidify the role of RatA in E. coli, but also demonstrate the importance of first validating the basics of molecular biology when investigating a previously poorly described gene, even in times of advanced high-throughput techniques. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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24 pages, 6117 KiB

Open AccessArticle

Functional Differentiation and Regulatory Mechanisms of Ferrochelatases HemH1 and HemH2 in Bacillus thuringiensis Under Iron and Oxidative Stress

by Jianghan Wang, Yi Luo, Tian Jiao, Shizhen Liu, Ting Liang, Huiting Mei, Shuang Cheng, Qian Yang, Jin He and Jianmei Su

Int. J. Mol. Sci. 2025, 26(7), 2911; https://doi.org/10.3390/ijms26072911 - 23 Mar 2025

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Abstract

Ferrochelatase is the terminal enzyme in heme biosynthesis. Bacillus thuringiensis (Bt) 97-27 contains two ferrochelatases, HemH1 and HemH2, but their regulatory mechanisms and functional differences under virous environmental stimuli remain unclear. This study confirmed that the iron uptake regulator protein (Fur) bound to [...] Read more.

Ferrochelatase is the terminal enzyme in heme biosynthesis. Bacillus thuringiensis (Bt) 97-27 contains two ferrochelatases, HemH1 and HemH2, but their regulatory mechanisms and functional differences under virous environmental stimuli remain unclear. This study confirmed that the iron uptake regulator protein (Fur) bound to the promoters of hemH1 and hemH2, with Fe²⁺ or Fe³⁺ enhancing this binding. Heterologous expression of HemH1 and HemH2 in Escherichia coli showed that pEH2/BL grew better than pEH1/BL under different 2,2′-Bipyridyl, Fe²⁺, and Fe³⁺ concentrations. Under iron limitation, the heme precursor ALA production decreased significantly in both strains. The heme production of pEH2/BL decreased sharply under iron-limited conditions, while that of pEH1/BL decreased significantly under iron-rich conditions. The H₂O₂ sensitivity experiment revealed that E. coli pEH1/BL was more tolerant to H₂O₂ than pEH2/BL. In Bt, ΔhemH2 was most sensitive to H₂O₂ stress, but complementation of hemH1 or hemH2 partially restored H₂O₂ resistance, with the overexpressed strain pHH2/Bt being most tolerant. β-galactosidase assays indicated that Fur positively regulated hemH1 and negatively regulated hemH2 under normal conditions, but this regulation reversed with 2.5 mM Fe³⁺. qRT-PCR showed upregulation of genes related to heme synthesis, oxidative stress, and ferrous iron transport. This study reveals the functional differentiation of HemH1 and HemH2 under the joint regulation of Fur and environmental factors, highlighting their synergistic roles in heme synthesis, heavy metal detoxification, and oxidative stress resistance to maintain bacterial physiological homeostasis. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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16 pages, 2241 KiB

Open AccessArticle

A Two-Phage Cocktail Modulates Gut Microbiota Composition and Metabolic Profiles in an Ex Vivo Colon Model

by Sthefhany Nohemí Rodríguez-Arellano, Jean Pierre González-Gómez, Bruno Gomez-Gil, Marisela González-Ávila, Juan Ramón Palomera-Hernández, Elisa Barrón-Cabrera, Marcela de Jesús Vergara-Jiménez and Cristobal Chaidez

Int. J. Mol. Sci. 2025, 26(6), 2805; https://doi.org/10.3390/ijms26062805 - 20 Mar 2025

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Abstract

Bacteriophage therapy is a promising approach for targeting antibiotic-resistant bacteria and modulating gut microbiota in metabolic diseases such as obesity. This study evaluated the impact of a two-phage cocktail on an ex vivo colonic simulation model of gut microbiota derived from obese individuals, [...] Read more.

Bacteriophage therapy is a promising approach for targeting antibiotic-resistant bacteria and modulating gut microbiota in metabolic diseases such as obesity. This study evaluated the impact of a two-phage cocktail on an ex vivo colonic simulation model of gut microbiota derived from obese individuals, both in its normalized state and after enrichment with Enterobacter cloacae, an obesity-related bacteria. Microbiological analyses confirmed that the phage cocktail remained active throughout the colonic regions over three digestion cycles and effectively reduced enterobacterial populations in the enriched microbiota. Metabarcoding of the 16S rRNA gene revealed that phage therapy did not significantly alter the abundance of dominant genera, but selectively reduced E. cloacae across all colonic regions. Alpha diversity was significantly affected only in the enriched microbiota, while beta diversity analysis indicated significant compositional shifts during therapy, with reduced dispersion in the final treatment stage. Short-chain fatty acid profiling demonstrated region- and group-specific metabolic responses, with increased lactic and butyric acid concentrations in the ascending colon of the enriched microbiota following phage treatment. This study provides the first ex vivo evidence that a two-phage cocktail can selectively eliminate E. cloacae while preserving overall microbiota structure and functionality. These findings establish a foundation for future in vivo studies exploring the role of phage therapy in reshaping gut microbial communities and metabolic profiles, highlighting its potential as a precision tool for managing gut dysbiosis in metabolic disorders. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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11 pages, 1931 KiB

Open AccessArticle

Detection of Live Shiga Toxin-Producing Escherichia coli with Long-Read Sequencing

by Katrina L. Counihan, Shannon Tilman, Chin-Yi Chen and Yiping He

Int. J. Mol. Sci. 2025, 26(5), 2228; https://doi.org/10.3390/ijms26052228 - 1 Mar 2025

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Abstract

A requirement of any foodborne pathogen testing method is that it only detects live bacteria. Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that penetrate the membranes of dead cells and form cross-linkages in the DNA, which prevents its amplification in PCR. [...] Read more.

A requirement of any foodborne pathogen testing method is that it only detects live bacteria. Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that penetrate the membranes of dead cells and form cross-linkages in the DNA, which prevents its amplification in PCR. This study investigated whether treatment with EMA or PMA would inhibit the sequencing of DNA from dead Escherichia coli. Range finding experiments with qPCR were conducted to determine the optimal concentrations of EMA and PMA needed to inhibit the amplification of DNA from dead cells while not influencing live cells. An EMA concentration that differentiated between live and dead cells could not be established. However, a PMA concentration of 25 µM effectively prevented qPCR amplification of DNA from dead E. coli while not impacting the amplification of live E. coli DNA. Sequencing experiments were conducted with PMA-treated live, untreated live, PMA-treated dead, and untreated dead E. coli. There were no significant differences in the detection of virulence genes of interest between the PMA-treated live, untreated live, and untreated dead E. coli. However, no DNA sequencing data were obtained from the PMA-treated dead E. coli. These results suggest that PMA could be incorporated into sample preparation methods prior to sequencing to selectively detect live cells of foodborne pathogens. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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11 pages, 3528 KiB

Open AccessArticle

N⁴-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs

by Jaunius Urbonavičius, Aušrinė Čekytė and Daiva Tauraitė

Int. J. Mol. Sci. 2025, 26(5), 1812; https://doi.org/10.3390/ijms26051812 - 20 Feb 2025

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Abstract

N⁴-methylcytosine is a modified heterocyclic base present both in RNA and DNA. The biosynthesis and function of this derivative are widely investigated. However, how the demethylation of this base occurs is not known. Here, we have investigated the growth of an [...] Read more.

N⁴-methylcytosine is a modified heterocyclic base present both in RNA and DNA. The biosynthesis and function of this derivative are widely investigated. However, how the demethylation of this base occurs is not known. Here, we have investigated the growth of an Escherichia coli uracil auxotroph strain in minimal M9 medium supplemented with N⁴-methylcytosine. We have found that this compound, but not the related N⁴,N⁴-dimethylcytosine, well supports growth with a generation time of the bacterium being 3 h compared to 1.5 h for media supplemented with cytosine or uracil. Using high-performance liquid chromatography (HPLC), we have demonstrated that the concentration of N⁴-methylcytosine in the growth medium decreases by 12% after 24 h of growth. We have shown that N⁴-methylcytosine is not directly converted into uracil by E. coli CodA cytosine deaminase. Instead, we propose the enzymatic pathway in which N⁴-methylcytosine is converted into cytosine by yet unknown demethylase, whereas CodA converts the resulting cytosine to uracil, thereby supporting the growth. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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12 pages, 2046 KiB

Open AccessArticle

Evidence for the Worldwide Distribution of a Bile Salt Hydrolase Gene in Enterococcus faecium Through Horizontal Gene Transfer

by Hiroyuki Kusada and Hideyuki Tamaki

Int. J. Mol. Sci. 2025, 26(2), 612; https://doi.org/10.3390/ijms26020612 - 13 Jan 2025

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Abstract

Bile salt hydrolase (BSH), a probiotic-related enzyme with cholesterol-assimilating and anti-hypercholesterolemic abilities, has been isolated from intestinal bacteria; however, BSH activity of bacteria in bile-salt-free (non-intestinal) environments is largely unknown. Here, we aimed to identify BSH from non-intestinal Enterococcus faecium and characterize its [...] Read more.

Bile salt hydrolase (BSH), a probiotic-related enzyme with cholesterol-assimilating and anti-hypercholesterolemic abilities, has been isolated from intestinal bacteria; however, BSH activity of bacteria in bile-salt-free (non-intestinal) environments is largely unknown. Here, we aimed to identify BSH from non-intestinal Enterococcus faecium and characterize its enzymatic function. We successfully isolated a plasmid-encoded bsh (efpBSH) from E. faecium, and the recombinant EfpBSH showed BSH activity that preferentially hydrolyzed taurine-conjugated bile salts, unlike the activity of known BSHs. EfpBSH functioned optimally at pH 4.0 and 50 °C. EfpBSH exhibited very low amino acid sequence similarity (48.46%) to EfBSH from E. faecalis T2 isolated from human urine, although 241 sequences with 100% identity to EfpBSH were found in both plasmids and chromosomes of E. faecium strains inhabiting intestinal and non-intestinal environments. Phylogenetically, EfpBSH was not affiliated with any known BSH phylogroup and was clearly distinguished from previously identified BSHs from intestinal lactic acid bacteria. Our genome database analysis demonstrated that horizontal gene transfer causes global efpBSH distribution among E. faecium strains in various environments (soil, water, and intestinal samples) and geographical regions (Asia, Africa, Europe, North America, South America, and Australia/Oceania). Overall, our findings are the first to indicate that BSH is not an intestine-specific enzyme and that hitherto-overlooked probiotic candidates with BSH activity can exist in diverse environments. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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24 pages, 5762 KiB

Open AccessArticle

Relative Distribution of DnaA and DNA in Escherichia coli Cells as a Factor of Their Phenotypic Variability

by Sharanya K. Namboodiri, Alexander Aranovich, Uzi Hadad, Levi A. Gheber, Mario Feingold and Itzhak Fishov

Int. J. Mol. Sci. 2025, 26(2), 464; https://doi.org/10.3390/ijms26020464 - 8 Jan 2025

Cited by 1 | Viewed by 959

Abstract

Phenotypic variability in isogenic bacterial populations is a remarkable feature that helps them cope with external stresses, yet it is incompletely understood. This variability can stem from gene expression noise and/or the unequal partitioning of low-copy-number freely diffusing proteins during cell division. Some [...] Read more.

Phenotypic variability in isogenic bacterial populations is a remarkable feature that helps them cope with external stresses, yet it is incompletely understood. This variability can stem from gene expression noise and/or the unequal partitioning of low-copy-number freely diffusing proteins during cell division. Some high-copy-number components are transiently associated with almost immobile large assemblies (hyperstructures) and may be unequally distributed, contributing to bacterial phenotypic variability. We focus on the nucleoid hyperstructure containing numerous DNA-associated proteins, including the replication initiator DnaA. Previously, we found an increasing asynchrony in the nucleoid segregation dynamics in growing E. coli cell lineages and suggested that variable replication initiation timing may be the main cause of this phenomenon. Here, we support this hypothesis revealing that DnaA/DNA variability represents a key factor leading to the enhanced asynchrony in E. coli. We followed the intra- and intercellular distribution of fluorescently tagged DnaA and histone-like HU chromosomally encoded under their native promoters. The diffusion rate of DnaA is low, corresponding to a diffusion-binding mode of mobility, but still one order faster than that of HU. The intracellular distribution of DnaA concentration is homogeneous in contrast to the significant asymmetry in the distribution of HU to the cell halves, leading to the unequal DNA content of nucleoids and DnaA/DNA ratios in future daughter compartments. Accordingly, the intercellular variabilities in HU concentration (CV = 26%) and DnaA/DNA ratio (CV = 18%) are high. The variable DnaA/DNA may cause a variable replication initiation time (initiation noise). Asynchronous initiation at different replication origins may, in turn, be the mechanism leading to the observed asymmetric intracellular DNA distribution. Our findings indicate that the feature determining the variability of the initiation time in E. coli is the DnaA/DNA ratio, rather than each of them separately. We provide a likely mechanism for the ‘loss of segregation synchrony’ phenomenon. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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14 pages, 1515 KiB

Open AccessArticle

Effect of Meropenem on Conjugative Plasmid Transfer in Klebsiella pneumoniae

by Daria A. Kondratieva, Julia R. Savelieva and Maria V. Golikova

Int. J. Mol. Sci. 2024, 25(23), 13193; https://doi.org/10.3390/ijms252313193 - 8 Dec 2024

Viewed by 1055

Abstract

Plasmid-mediated resistance is a major mechanism that contributes to the gradual decline in the effectiveness of antibiotics from different classes, including carbapenems. Antibiotics can significantly contribute to the efficiency of plasmid transfer between bacterial strains. To investigate the potential effect of an antibiotic [...] Read more.

Plasmid-mediated resistance is a major mechanism that contributes to the gradual decline in the effectiveness of antibiotics from different classes, including carbapenems. Antibiotics can significantly contribute to the efficiency of plasmid transfer between bacterial strains. To investigate the potential effect of an antibiotic on the efficacy of conjugative plasmid transfer, we conducted mating experiments with Klebsiella pneumoniae strains. Donor strains of K. pneumoniae that carry plasmids with bla_KPC or bla_OXA-48 carbapenemase genes and recipient plasmid-free K. pneumoniae strains were used in matings. Matings were conducted on the agar with or without meropenem at 1/8×, 1/4×, or 1/2×MIC against the respective recipients. In the second part of our study, we investigated the pharmacodynamic properties of meropenem against transconjugant strains of K. pneumoniae, which were obtained in the first part of this study. As a result, at a concentration equivalent to 1/8×MIC, meropenem primarily inhibited conjugation among K. pneumoniae strains, while at a concentration equal to 1/2×MIC, it facilitated conjugation. Transconjugants derived from K. pneumoniae with intermediate MICs failed to respond to simulated treatment with meropenem using prolonged infusion and a high-dose regimen. This finding suggests that such transconjugants may potentially pose a risk if involved in an infectious process. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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Review

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27 pages, 1094 KiB

Open AccessReview

Assessing Salmonella Typhi Pathogenicity and Prevention: The Crucial Role of Vaccination in Combating Typhoid Fever

by Elena Roxana Buzilă, Olivia Simona Dorneanu, Felicia Trofin, Cristina Mihaela Sima and Luminița Smaranda Iancu

Int. J. Mol. Sci. 2025, 26(9), 3981; https://doi.org/10.3390/ijms26093981 - 23 Apr 2025

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Abstract

Enteric fever is caused by Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi (S. Paratyphi) A, B, and C. Globally, an estimated 11 to 21 million cases of typhoid and paratyphoid fever occur annually, with approximately 130,000–160,000 deaths, [...] Read more.

Enteric fever is caused by Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi (S. Paratyphi) A, B, and C. Globally, an estimated 11 to 21 million cases of typhoid and paratyphoid fever occur annually, with approximately 130,000–160,000 deaths, most of which are reported in South/Southeast Asia and sub-Saharan Africa. The antibiotic susceptibility of S. Typhi strains varies between countries within broad limits, from 3% to 97% for ampicillin, 9% to 95% for ciprofloxacin, 4% to 94% for chloramphenicol (India vs. Pakistan), and 0% to 99% for ceftriaxone (India vs. Iraq). With S. Typhi increasingly exhibiting resistance to antibiotics, vaccination becomes an essential preventive measure. Currently, three vaccines are licensed for typhoid fever: the typhoid conjugate vaccine (TCV), live-attenuated oral vaccine Ty21a (Ty21a), and Vi capsular polysaccharide vaccine (Vi-CPS). While no specific vaccine exists for paratyphoid fever, the genetic and antigenic similarities between S. Paratyphi and S. Typhi offer potential for the development of such a vaccine. Early studies show promising results, demonstrating both safety and immunogenicity in preclinical trials. Whole genome sequencing (WGS) provides a powerful tool for assigning genotypes, identifying plasmids, comparing genetic elements, and investigating molecular factors that contribute to antibiotic resistance and virulence. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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22 pages, 366 KiB

Open AccessReview

Salmonella Type III Secretion System Effectors

by Micah J. Worley

Int. J. Mol. Sci. 2025, 26(6), 2611; https://doi.org/10.3390/ijms26062611 - 14 Mar 2025

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Abstract

Salmonella is estimated to infect between 200 million and over 1 billion people per year. The exact number is not known, as many cases go unreported. Integral to the pathogenesis of Salmonella, as well as numerous other Gram-negative pathogens, is its type [...] Read more.

Salmonella is estimated to infect between 200 million and over 1 billion people per year. The exact number is not known, as many cases go unreported. Integral to the pathogenesis of Salmonella, as well as numerous other Gram-negative pathogens, is its type III effectors. Salmonella possesses two distinct type III secretion systems, encoded by Salmonella pathogenicity island-1 and Salmonella pathogenicity island-2. Together, they secrete at least 49 type III effectors into host cells that are collectively responsible for many of the virulence attributes of this pathogen. These virulence factors facilitate the invasion of host cells, induce and attenuate inflammation, and change the migratory properties of infected phagocytes, among other things. The effects of all type III effectors on Salmonella virulence are discussed. Full article

(This article belongs to the Special Issue Molecular Research on Bacteria)

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