Transformation of foreign DNA, subjecting Escherichia coli to CaCl2 treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its popularity in E. coli, heat shock transformation is rarely reported in other Gram-negative bacteria. The objective of this research is to demonstrate that the heat shock transformation method can also be performed to introduce genes in different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida, and Enterobacter roggenkampii. A green fluorescent plasmid, pDSK-GFPuv, having the size of 8.9 kb, was directly transferred to R. pseudosolanacearum by heat shock at 50 °C for 60s. For P. aeruginosa, P. putida, and E. roggenkampii, the cells were made competent using CaCl2, followed by heat shock transformation at 50 °C for 180s. As the plasmid contains a gene encoding green fluorescence protein, after successful transformation, bacterial strains were demonstrated for colonization in tomato seedlings. The introduced plasmid was re-isolated from R. pseudosolanacearum to prove its stability inside the bacterium after the heat shock transformation. This easy and convenient method of transformation may be used for other Gram-negative bacteria to prove its wider applicability.

Abstract Ralstonia pseudosolanacearum ( Rps ) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 10 9 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB:: Ω mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA:: Ω. It indicated that the necrotic lesion caused in tomato leaves was hrp -dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

Abstract Mutation in genomes is mainly attributed to replication, though transcription is also known to be mutagenic and is a more frequent event than replication in an organism. Recently, there have been reports regarding genome-wide transcription-induced mutagenesis. However, a distinct demonstration of specific mutation being replication-dependent and/or transcription-dependent in genomes is yet to be established. Here, we studied synonymous single-nucleotide polymorphisms (SNPs) in 2091 individual coding sequences (CDS) in the leading strand (LeS) and the lagging strand (LaS) of the Escherichia coli chromosome by comparing across 157 strains. The frequencies of complementary transitions ( ti ) and complementary transversions ( tv ) were compared in each CDS to assess parity violation in the context of strand location and gene expression. The C→T and G→A exhibited the maximum frequency as well as the most prominent strand inequality as these tis were influenced both by the strand location as well as by the gene expression. Interestingly, inequality between T→C and A→G was expression-dependent but strand-independent. This was a direct demonstration of strand inequality due to expression but not due to replication. A→T and G→T tv s were universally more frequent than their complementary T→A and C→A, respectively. The strand-independent but expression-dependent synonymous SNP inequality in CDS, supports the role of transcription-induced mutagenesis contributing to strand inequality in the E. coli chromosome. Significance Statement Mutational patterns in bacterial genomes are shaped by both replication and transcription, but their relative roles remain unclear. By analyzing synonymous SNPs across 157 strains of E. coli , we identify strong strand-specific asymmetry consistent with replication-associated biases. A strong asymmetry between the strands regarding C→T and G→A ti is observed, as it is influenced by replication asymmetry and expression. We further show that gene expression significantly influences mutation types, with T→C ti being enriched in the highly expressed genes and A→G ti being enriched in the lowly expressed genes. Notably, A→T ti is strand independent but exhibits mild dependency on gene expression, revealing a previously unrecognized mutational pattern. These findings highlight how some SNPs are influenced by transcription but not replication

Ralstonia pseudosolanacearum F1C1 is a soil-borne phytopathogenic bacterium with a broad host range that infects several economically important crops. This study primarily focuses on the infection of this phytopathogen in two such important crop seedlings: tomato and eggplant. The observations of the study reveal a complex set of symptoms that include drooping and blackening of the seedling stem, as well as blackening, chlorosis, and curling of cotyledon leaves. Notably, the symptom of stem softening and translucency is seen primarily in the water-submerged stem regions of both root- and leaf-inoculated seedlings. While the wild-type R. pseudosolanacearum F1C1 strain and its weakly virulent mutant phcA::Ω exhibited this phenotype in both tomato and eggplant seedlings, the virulence-deficient hrpB::Ω mutant did so only in a few eggplant seedlings but not in tomato seedlings. By investigating these unique pathological phenotypes in the infected seedlings, this study uncovers a unique symptom previously not reported in seedling inoculation experiments. The work also touches upon the distinct escape mechanisms exhibited by seedlings, revealing how some of its host plants might resist wilting despite infection, offering new avenues for future research. These findings contribute to the understanding of R. pseudosolanacearum pathogenesis, shedding light on its virulence and host response mechanisms.

The three stop codons TAA, TGA and TAG are not used equally for translation termination in organisms. Assuming these codons to be synonymous, we computed relative synonymous codon usage (RSCU) values among protein-coding genes in the Escherichia coli genome. TAA showed a high RSCU value (1.941), nearly twice the expected value (1.0), whereas TGA (0.851) was near expected and TAG (0.208) was strongly avoided. Genes with high codon adaptation index (CAI) values exhibited significantly higher TAA usage (RSCU 2.558) than genes with low CAI values (1.790), suggesting stronger selection on TAA for optimal gene expression. Analysis of genes with experimentally estimated expression levels further supported selection favoring TAA. Operon-wise analysis of co-transcribed genes in the E. coli genome revealed that highly expressed operons showed a strong preference for the TAA stop codon, whereas moderately and weakly expressed operons exhibited mixed stop codon usage patterns. Pearson correlation analysis demonstrated a significant positive association between TAA usage and average operon expression level (r = 0.66, p = 0.0367), suggesting selective pressure favoring TAA-mediated efficient translation termination in highly expressed bacterial operons. This selection on TAA codon is found to be similar among genes in the leading and lagging strands of the DNA replication fork suggesting strand independence nature of selection pressure. Collectively, this study presents a model on stop codon selection, wherein termination optimization follows the order TAA > TGA>TAG. These results provides new insights for understanding evolution of coding sequences at molecular level.

Ralstonia solanacearum, a gram-negative bacterium, is known to cause devastating wilting disease in many plant species. The bacterium's pathogenesis relies on various virulence determinants, including twitching motility, facilitated by type IV pili appendages. Twitching motility is a density-dependent mechanism enabling bacterial movement on solid surfaces, crucial for biofilm formation and virulence. Understanding this motility is of utmost importance in studying the pathogen's behavior. In this chapter, we present a simple and reproducible method to assess twitching motility in R. solanacearum. The method involves microliter spotting of a diluted bacterial suspension onto a solid agar growth medium, followed by microscopic observation of the early-stage microcolonies. Twitching motility becomes evident as finger-like projections, which merge and progressively spread in all directions, forming multilayered structures. The examination also reveals cell heterogeneity within colonies and the influence of bacterial concentration on microcolony shape, potentially attributed to quorum sensing. Our simplified protocol for assessing twitching motility in microcolonies using microliter spotting offers significant advantages over existing methods described in the literature, making it an effective tool for investigating twitching motility in bacterial pathogenesis. A detailed step-by-step description of the protocol is provided in this chapter, facilitating its adoption and application in future research.

The increasing availability of large-scale variants datasets have enabled systematic in silico analyses of mutation patterns in different disease-associated genes. In this study, we have performed a comprehensive computational analysis of BRCA1 variants spanning intronic, coding, and non-coding regions using publicly available repositories. The BRCA1 gene, which is essential for tumor suppression function becomes a major driver of hereditary breast and ovarian cancer (HBOC). A domain-specific nucleotide-level mutational trend analyses will provide a useful framework for translational research. Our study revealed a predominance of GàT and CàA transversions across BRCA1 variants, with notable enrichment for specific predicted impact categories. Our domain-specific analyses revealed that the RING domain (exons 2-7) and BRCT domain (exons 17-22), known mutation hotspots, frequently exhibited GàT and GàC transversions. The PolyPhen-based classification indicated that a substantial proportion of variants fall into higher predicted impact groups, which exhibited distinct substitution biases compared to benign categories. AI-assisted computational analyses using SpliceAI and RegulomeDB indicated predominantly moderate to benign splicing effects among intronic variants, while a subset showed potential regulatory impact that requires validation in large-scale studies. To our observation, BRCA1 possessed a balanced codon usage with high Nc and Nc' values. Furthermore, we highlight BRCA1 evolutionary and mutational patterns, particularly substitution trends, at nucleotide level. Therefore, the study offers a computational framework for prioritizing variants through AI-enabled tools and substitution patterns for experimental validation and functional characterization.

Transformation of foreign DNA, subjecting Escherichia coli to CaCl2 treatment followed by heat shock exposure, is a regular approach in recombinant DNA technology. However, in spite of its large popularity in E. coli , heat shock transformation is rarely reported in other Gram-negative bacteria. Instead, techniques such as natural transformation, conjugation, or electroporation are used in those bacteria. In this study, we have successfully implemented the heat shock transformation in four different Gram-negative bacteria, such as Ralstonia pseudosolanacearum, Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii . The standard heat shock transformation procedure used for E. coli DH5α has been modified. The pDSK-GFPuv plasmid bearing the green fluorescence gene was directly transferred to Ralstonia pseudosolanacearum by heat shock at 50 ºC for 60 seconds. For Pseudomonas aeruginosa, Pseudomonas putida , and Enterobacter roggenkampii , the cells were made competent using CaCl2, followed by performing transformation by heat shock at 50 ºC for 180 seconds. The transformants were resistant to kanamycin as well as exhibited fluorescence. These transformants were used to study the colonization pattern of tomato seedlings. Our study suggested that the heat shock transformation method can also be performed to introduce genes in other Gram-negative bacteria, other than E. coli . ### Competing Interest Statement The authors have declared no competing interest.

Rotavirus (RV) is a major aetiology of childhood gastroenteritis worldwide. It is crucial to understand the hospital-based RV disease prevalence and its spatio-temporal genotype distribution during the period pre-and post-introduction of RV vaccines in India. A systematic review and meta-analysis were performed to extract information on literature related to the impact of vaccination on rotavirus disease prevalence and the distribution of genotypes from 1986 to 2022. A search for relevant articles was carried out in public databases (PubMed, Google Scholar, and ScienceDirect) to extract specific information on RV prevalence among children less than 5 years of age and the genotype distribution from 1986 to 2022. DerSimonian-Laird random-effects model was employed to account for the heterogeneity of included studies analysed using meta-analysis and publication bias was assessed using funnel plot and Egger linear regression test. Of the 1939 records identified through screening and after removing duplicate records, the full texts of 1609 records were assessed for eligibility. After the full-text assessment, 49 records were found eligible and included in the study. The estimated pooled prevalence of RV-associated gastroenteritis during the pre-vaccine period was 33% {(95% confidence interval (CI), 28%-38%)} while the prevalence was 23% (95% CI, 18%-29%) in the post-vaccine period. Rotaviruses are classified into genotypes by their G-(glycoprotein VP7) and P-(protease-sensitive VP4) proteins. Combination of genotype G1 and P[8] that is G1P[8] predominated during the pre- and post-vaccine period, while the prevalence of G3P[8] increased after immunisation. The dominant genotypes in pre-vaccine era were G1 and G2 while G1 and G3 after vaccine inclusion, with a constant circulation of P[8] during the entire period from 1986 to 2022. Occurence of G2 increased post-vaccination in western zone of India. As observed from the meta-regression analysis, rotavirus vaccination has significantly reduced gastroenteritis associated hospitalizations and death. The spatio-temporal change in the genotype distribution in the post-vaccination era warrants the need for further surveillance studies to provide information on RV-associated hospital visits. Additionally, this will also provide information on detection of emerging strains that can assist in designing future policies for the implementation and development of new-generation vaccines against rotavirus disease.

Influenza viruses are a leading cause of severe respiratory illness worldwide, with growing resistance to M2 and neuraminidase inhibitors emphasizing the urgent need for new antiviral target. As Antivirals targeting RNA polymerase are less susceptible to resistance, there is increasing interest in drugs that inhibit RdRp. This study evaluates the inhibitory potential of phytochemicals derived from Citrus limon(Lemon) against influenza RdRp. Two PB2 proteins of influenza H3N2 (PDB ID: 4p1u) and H1N1 (PDB ID: 7as0) subunits were considered for this study Conducting molecular docking of lemon metabolites with these target proteins. Based on binding affinity, six phytocompounds were selected for in-silico ADMET analysis which demonstrated acceptable physiochemical, pharmacokinetic and non-toxic profiles. The top five PB2-ligand complexes were further analyzed via molecular dynamics (MD) simulation. MD simulation results revealed that apigenin, luteolin, and quercetin exhibited stable interactions with both proteins. The binding free energy and contributed energies were calculated by using the MM-GBSA method. QM/MM calculations on the lowest-energy complexes suggested that these phytocompounds may effectively block the PB2 cap-binding domain. Thus, lemon-derived natural compounds hold promising as anti-influenza agent, offering a pathway for drug development to address the clinical challenges posed by influenza.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plant species, posing significant economic challenges. Although tomato has been a primary model host for investigating the pathogenicity and systemic infection of this bacterium, this manuscript presents a comparative pathogenicity study between two closely related solanaceous hosts, tomato and eggplant, revealing differential host responses to the same pathogen, specifically through leaves and roots. Interestingly, eggplant seedlings exhibited a significantly higher susceptibility to cotyledon leaf inoculation than tomato seedlings. In the case of leaf inoculations, a few tomato seedlings escaping wilting (called escapees) were a usual observation at a high pathogen load (109 CFU/mL); but in the case of eggplant seedlings, no escapees were observed even at a 100-fold lower pathogen concentration. The greater susceptibility of eggplant was further demonstrated by performing both single- and double-leaf inoculations in the seedlings. Surprisingly, root inoculations resulted in a significantly lower wilting for eggplant than for tomato seedlings. Interestingly, the escapees and wilted seedlings of tomato and eggplant were found to harbour R. pseudosolanacearum F1C1. The contrasting susceptibility between the two hosts regarding root and leaf regions indicates the tissue-dependent nature of susceptibility. The study underscores the value of employing multiple host species to uncover new insights into pathogen behaviour and host-pathogen interactions.

Plant-associated microbiome plays important role in maintaining overall health of the host plant. Xanthium strumarium displaying resilience to various environmental fluctuations may harbor some bacterial isolates which can help this plant to grow worldwide. The present study aims to isolate endophytic and rhizospheric bacteria from X. strumarium and assess their plant growth-promoting and Ralstonia solanacearum antagonism activity. From a total of 148 isolated bacteria, 7 endophytic and 2 rhizospheric bacterial isolates were found to endow with significant in vitro plant growth promotion activities. The 16S rRNA gene sequence similarity of the 7 endophytic isolates has revealed these bacteria belonging to 5 genera viz. Curtobacterium, Pantoea, Pseudomonas, Microbacterium and Paracoccus whereas, the two rhizospheric isolates were identified as species of Ralstonia pickettii and Priestia megaterium. Maximum growth promotion was observed using the strains Pseudomonas fluorescens XSS6 and Microbacterium hydrothermale XSS20 in the assay conducted on tomato plants. In the in planta inhibition assay of R. solanacearum carried out in tomato seedlings using root bacterization method, Pseudomonas fluorescens XSS6 and Panotea vagans XSS3 showed antagonistic activity with biocontrol efficacy of 94.83% and 83.96%, respectively. GC-MS analysis detected several known antimicrobial compounds in the extract of the culture supernatant of Pseudomonas fluorescens XSS6 and Panotea vagans XSS3 strains, which may contribute to the inhibition of R. solanacearum by these strains. The results of our study indicated that the bacteria associated with X. strumarium exhibit multiple plant-beneficial effects. These bacteria have the potential to be developed as effective biofertilizers and biological control agents, promoting sustainable agriculture practices.

Endophytes are a class of non-pathogenic microorganisms that reside within a plant and contribute to their health. Here, we isolated several endophytes from cotyledon-stage tomato seedlings that inhibited the growth of Ralstonia pseudosolanacearum , the bacterial wilt pathogen of tomato. One such endophyte, Pseudomonas aeruginosa SPT08, protected tomato seedlings as well as grown-up plants from the wilt disease. SPT08 also improved the tomato plant height by 20% and root growth by 60% in weight. SPT08 colonization inside tomato seedlings as well as in grown-up plants was studied using green fluorescent protein to demonstrate its endophytic behavior. SPT08 exhibited twitching and swimming motility and produced extracellular enzymes such as pectinase, protease, and amylase. SPT08 tested positive for several plant growth-promoting features such as phosphate solubilization, production of siderophore, plant hormone auxin, hydrogen cyanide, and ammonia. These features were further corroborated with SPT08 whole genome sequence. SPT08 genome is 6265489 bp, with 66.59% G+C and 5786 coding genes, including type II, III, and VI protein secretion systems. The antiSMASH tool identified potential for several secondary metabolites, including antibiotics, in SPT08. This study underscores the utility of P. aeruginosa SPT08 bio-protection from bacterial wilt and growth promotion agent in tomato. Highlights Pseudomonas aeruginosa SPT08, an endophyte isolated from tomato seedlings that inhibits bacterial wilt pathogen R. pseudosolanacearum, can control bacterial wilt in tomato plants and enhance the height and root growth. ### Competing Interest Statement The authors have declared no competing interest.

The genome G+C content of bacteria varies widely, from 13% to 75%, which is influenced by both environmental and internal mutation pressure; however, the precise determinants of this variability remain unresolved. Mutation-based models, such as Sueoka’s directional mutation hypothesis, suggest that G+C content arises from mutational pressures within an organism without providing any specific advantage to it. Though there are several advantages associated with genome G+C%, there is limited evidence, favoring any selection mechanism for G+C% evolution. Hence, the genome G+C% in organisms is largely studied under the neutral theory of evolution. Cytosine deamination and guanine oxidation are recognized as major contributors to A/T mutational bias, producing frequent substitutions such as C→T transitions and G→T transversions, respectively. While these mechanisms leading to A+T enrichment have been well studied, counteracting processes that promote G+C enrichment in organisms are comparatively less understood. This review mainly highlights adenine as an underexplored contributor: its deamination and oxidation yield A→G and A→C substitutions, respectively, both biased toward increased G+C content. We further consider how the efficiency of DNA repair mechanisms may shape G+C content across evolutionary timescales. Together, these perspectives address a gap in the current understanding of the mutational forces influencing genome composition.

Background: How parents interact with their children and what they are exposed to can have a big impact on a child's language development. Nowadays, videos on different devices are an important source of information and stimulation for kids from a young age. Many researchers have studied how screen time may affect a child's language development. Objectives: The purpose of this review was to explore the available research on the relationship between screen time and language development in children under 12. Methods: This scoping review involved a systematic search of the database using predefined criteria for available research regarding the impact of screen time on language development in children younger than 12 years. Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for Scoping Reviews (PRISMA-ScR) framework was adhered to for this review. Researchers did a qualitative and thematic analysis of the included research. Any conflict of opinion was resolved by discussion. Results: 16 studies were selected for this review, of which nine reported a negative impact of screen time on language development, five reported no significant impact and two reported a positive effect. Conclusions: After reviewing various studies, it has been found that increased screen time can have a negative impact on a child's language development. While some studies show no impact or even a positive impact, factors such as the duration of viewing, video characteristics, content, and co-viewing with adults also play a role. Overall, it seems that the negative effects of screen time outweigh the positive ones.

Antisense medications treat diseases that cannot be treated using traditional pharmacological technologies. Nucleotide monomers of bare and phosphorothioate (PS)-modified LNA, N-MeO-amino-BNA, 2',4'-BNANC[NH], 2',4'-BNANC[NMe], and N-Me-aminooxy-BNA antisense modifications were considered for a detailed DFT-based quantum chemical study to estimate their molecular-level structural and electronic properties. Oligomer hybrid duplex stability is described by performing an elaborate MD simulation study by incorporating the PS-LNA and PS-BNA antisense modifications onto 14-mer ASO/RNA hybrid gapmer type duplexes targeting protein PTEN mRNA nucleic acid sequence (5'-CTTAGCACTGGCCT-3'/3'-GAAUCGUGACCGGA-5'). Replica sets of MD simulations were performed accounting to two data sets, each set simulated for 1 μs simulation time. Bulk properties of oligomers are regulated by the chemical properties of their monomers. As such, the primary goal of this work focused on establishing an organized connection between the monomeric BNA nucleotide's electronic effects observed in DFT studies and the macroscopic behavior of the BNA antisense oligomers, as observed in MD simulations. The results from this study predicted that spatial orientation of MO-isosurfaces of the BNA nucleotides are concentrated in the nucleobase region. These BNA nucleotides may become less accessible for various electronic interactions when coupled as ASOs forming duplexes with target RNAs and when the ASO/RNA duplexes further bind with the RNase H. Understanding such electronic interactions is crucial to design superior antisense modifications with specific electronic properties. Also, for the particular nucleic acid sequence solvation of the duplexes although were higher compared to the natural oligonucleotides, their binding energies being relatively lower may lead to decreased antisense activity compared to existing analogs such as the LNAs and MOEs. Fine tuning these BNAs to obtain superior binding affinity is thus a necessity.

Bacteriological sampling by dilution plating is an age old fundamental microbiological procedure being performed for enumerating viable cells. Serial dilution approach serves an integral part of it wherein a given fold dilution can be attained by stepwise dilutions using different sample volumes as follows: a 106 fold dilution can be attained by successively relaying 100 μL six times or 10 μL three times or 1 μL two times to a final 1 mL volume each time in the respective 10-, 100- and 1000-fold dilution series. Considering the random nature of bacterial distribution in culture, relaying of a higher sample volume (as 100 μL in 10-fold series) seems more accurate; however when complexity of the process (defined by number of steps involved) is considered, relaying of a lower sample volume (as 1 μL in 1000-fold series) seems more accurate. Considering the opposite relationship between sample volume and step complexity involved, it is pertinent to find out the better approach for CFU estimation. Besides this, as serial dilution can be performed using different diluent volumes in order to adjust to a certain final volume such as 1 mL or 10 mL, it is also pertinent to observe the impact of diluent volume on CFU estimation. Therefore, the current study was undertaken to understand the impact of two variable factors of serial dilution: (i) fold dilution and (ii) diluent volume being used on the CFU estimation of bacteria by carrying out a critical examination of 10-, 100- and 1000-fold dilution series. The studies were conducted using two different bacterial cell suspensions: Escherichia coli DH5α and Ralstonia pseudosolanacearum F1C1 exhibiting two different colony morphologies. The results revealed that bacterial CFU estimation is indifferent to the fold dilution as well as the diluent volume being used. Overall, the study addresses different fundamental yet unexplored aspects of bacterial sampling. ### Competing Interest Statement The authors have declared no competing interest.

Base substitution mutations such as transition (ti) and transversion (tv) in organisms are major driving force in molecular evolution. In this study, different possible types of base pairing that can cause ti and tv were investigated using the density functional theory (DFT) method. The chemical structures of bases as well as base pairs were optimized using B3LYP hybrid functional along with 6-31G(d,p) basis set. We performed single point energy calculation of all optimized species using the same functional but combined with higher diffuse and polarized basis set i.e. 6-311++G(d,p) to get more refined energy of all species. The binding energy of various base pairs was calculated considering basis set superposition error (BSSE) as well as without BSSE. The binding energy of the base pairs leading to ti were found to be more stable than that of the base pairs leading to tv. This was interesting considering the observations in organisms that tis are more frequent than tvs. Among the base pairs leading to the same ti, G(keto): T (enol) was found to be more stable than A(imino):C(amino) base pairs. This theoretical study of binding energy of different base pairs using the DFT method has provided additional evidences in support to the biological observations of a higher transition rate than transversion in genomes. ### Competing Interest Statement The authors have declared no competing interest.

Codon usage bias (CUB), the uneven usage of synonymous codons encoding the same amino acid, differs among genes within and across bacteria genomes. CUB is known to be influenced by gene expression and accordingly, CUB differs between the high-expression and low-expression genes in several bacteria. In this article, we have extended codon usage study considering gene essentiality as a feature. Using machine learning (ML) based approaches, we have analysed Relative Synonymous Codon Usage (RSCU) values between essential and non-essential genes in Escherichia coli and thirty-four other bacterial genomes whose gene essentiality features were available in public databases. We observed significant differences in codon usage patterns between essential and non-essential genes for majority of the bacterial genomes and accordingly, ML based classifiers achieved high area under curve (AUC) scores, with a minimum score of 70.0 across twenty-eight organisms. Further, importance of the codons towards classifying genes found to differ among the codons in each genome. Arg codon CGT and Gly codon GGT were observed to be the most preferred codons among essential genes in Escherichia coli. Interestingly, some of the codons like CGT, ATA, GGT and GGG observed to be contributing consistently towards classifying essential genes across thirty-five bacteria genomes studied. In other hand, codons TGY and CAY encoding amino acids Cys and His respectively were among the least contributing codons towards classification among all these bacteria. This study demonstrates the gene essentiality based differences in synonymous codon usage in bacteria genomes and presents a common codon usage pattern across bacteria.

For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFUs) has always been the preferred method in microbiology owing to its simplicity, albeit being laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing micro-colonies in conjunction with the benefits of a microscope. Here, we employed a simple method of five to ten microliter spotting of a diluted bacterial culture multiple times on a single Petri dish followed by determining CFU by counting micro-colonies using a phase -contrast microscope. In this method, the CFU of an Escherichia coli culture can be estimated within a four-hour period after spotting. Further, within a ten-hour period after spotting, CFU in a culture of Ralstonia sol-anacearum, a bacterium with a generation time of around 2 h, can be estimated. The CFU number determined by micro-colonies observed for 106-fold dilutions or lower is similar to that obtained by the dilution plating method for 107-fold dilutions or lower. Micro-colony numbers observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later hours (4 h in case of E. coli and 10 h in case of R. solanacearum), where the visibility of the colonies was better due to a noticeable increase in the size of the colonies. This suggested that micro-colonies observed in the early hours indeed represent the bacterial number in the culture. Practical applications to this counting method were employed in studying the rifampicin-resistant mutation rate as well as performing a fluctuation test in E. coli. The spotting method described here to enumerate bacterial CFU results in reduction of labour, time and resources.