scholarly journals Fluorescently Labeled DNA Interacts with Competence and Recombination Proteins and Is Integrated and Expressed Following Natural Transformation ofBacillus subtilis

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Mirjam Boonstra ◽  
Nina Vesel ◽  
Oscar P. Kuipers
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Bacterial Species ◽  
Time Lapse ◽  
Transformation Process ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Recombination Proteins ◽  
Homologous Locus ◽  
Fluorescent Nucleotides

ABSTRACTDuring competence,Bacillus subtilisis able to take up DNA from its environment through the process of transformation. We investigated the ability ofB. subtilisto take up fluorescently labeled DNA and found that it is able to take up fluorescein-dUTP-, DyLight 550-dUTP-, and DyLight 650-dUTP-labeled DNA. Transformation with labeled DNA containing an antibiotic cassette resulted in uptake of the labeled DNA and also generated antibiotic-resistant colonies. DNA is primarily taken up at the pole, as it can be seen to colocalize with ComFC, which is a component of the competence machinery. The DNA is taken up rapidly and can be seen to localize with (the actively searching form of) RecA. Colocalization with a homologous locus on the chromosome increases over time. Using microfluidics, we observed replacement of the homologous locus and subsequent expression of the integrated labeled and unlabeled DNA, although whether the integrated DNA contains labeled nucleotides needs to be determined conclusively. Integrated DNA in cells with a doubling time of 60 min is expressed on average 6 h 45 min after the addition of DNA and 4 h 45 min after the addition of fresh medium. We also found that the expression of the incoming DNA under these conditions can occur before cell division and, thus, before complete exit from the competence state. Because the competence machinery is conserved among naturally competent bacteria, this method of labeling is also suitable for studying transformation of other naturally competent bacteria.IMPORTANCEWe used DNA that was covalently labeled with fluorescent nucleotides to investigate the transformation process ofBacillus subtilisat the molecular level. We show that the labeled DNA colocalizes with components of the competence machinery, the chromosome, and the recombination protein RecA. Using time-lapse microscopy and microfluidics, we visualized, in real-time, the uptake of fluorescently labeled DNA. We found that under these conditions, cell division is not required for the expression of integrated DNA. Because the competence machinery is conserved in naturally competent bacteria, this method can also be used to investigate the transformation process in many other bacterial species.

scholarly journals Noc Corrals Migration of FtsZ Protofilaments during Cytokinesis in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuanchen Yu ◽  
Jinsheng Zhou ◽  
Frederico J. Gueiros-Filho ◽  
Daniel B. Kearns ◽  
Stephen C. Jacobson
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescence Microscopy ◽  
Control Cell ◽  
Time Lapse ◽  
Ring Formation ◽  
Microfluidic Channels ◽  
Content Type ◽  
The Future ◽  
Z Ring

ABSTRACT Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In Bacillus subtilis, the DNA- and membrane-binding Noc protein is thought to participate in nucleoid occlusion by preventing FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent de novo FtsZ ring formation over the chromosome nor does Noc control cell division site selection. Instead, Noc corrals FtsZ at the cytokinetic ring and reduces migration of protofilaments over the chromosome to the future site of cell division. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the diffuse FtsZ rings observed in the Noc mutant can be suppressed by ZapA overexpression. Thus, Noc sterically hinders FtsZ migration away from the Z-ring during cytokinesis and retains FtsZ at the postdivisional polar site for full disassembly by the Min system. IMPORTANCE In bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery at the midcell, and Z-ring formation is discouraged over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated, at least in part, by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

scholarly journals Cellular Architecture Mediates DivIVA Ultrastructure and Regulates Min Activity in Bacillus subtilis

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Prahathees Eswaramoorthy ◽  
Marcella L. Erb ◽  
James A. Gregory ◽  
Jared Silverman ◽  
Kit Pogliano ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Spatial Organization ◽  
Time Lapse ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Content Type ◽  
Temporal Regulation ◽  
Min Proteins

ABSTRACTThe assembly of the cell division machinery at midcell is a critical step of cytokinesis. Many rod-shaped bacteria position septa using nucleoid occlusion, which prevents division over the chromosome, and the Min system, which prevents division near the poles. Here we examined thein vivoassembly of theBacillus subtilisMinCD targeting proteins DivIVA, a peripheral membrane protein that preferentially localizes to negatively curved membranes and resembles eukaryotic tropomyosins, and MinJ, which recruits MinCD to DivIVA. We used structured illumination microscopy to demonstrate that both DivIVA and MinJ localize as double rings that flank the septum and first appear early in septal biosynthesis. The subsequent recruitment of MinCD to these double rings would separate the Min proteins from their target, FtsZ, spatially regulating Min activity and allowing continued cell division. Curvature-based localization would also provide temporal regulation, since DivIVA and the Min proteins would localize to midcell after the onset of division. We use time-lapse microscopy and fluorescence recovery after photobleaching to demonstrate that DivIVA rings are highly stable and are constructed from newly synthesized DivIVA molecules. After cell division, DivIVA rings appear to collapse into patches at the rounded cell poles of separated cells, with little or no incorporation of newly synthesized subunits. Thus, changes in cell architecture mediate both the initial recruitment of DivIVA to sites of cell division and the subsequent collapse of these rings into patches (or rings of smaller diameter), while curvature-based localization of DivIVA spatially and temporally regulates Min activity.IMPORTANCEThe Min systems ofEscherichia coliandBacillus subtilisboth inhibit FtsZ assembly, but one key difference between these two species is that whereas theE. coliMin proteins localize to the poles, theB. subtilisproteins localize to nascent division sites by interaction with DivIVA and MinJ. It is unclear how MinC activity at midcell is regulated to prevent it from interfering with FtsZ engaged in medial cell division. We used superresolution microscopy to demonstrate that DivIVA and MinJ, which localize MinCD, assemble double rings that flank active division sites and septa. This curvature-based localization mechanism holds MinCD away from the FtsZ ring at midcell, and we propose that this spatial organization is the primary mechanism by which MinC activity is regulated to allow division at midcell. Curvature-based localization also conveys temporal regulation, since it ensures that MinC localizes after the onset of division.

scholarly journals Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Robert S. Brzozowski ◽  
Brooke R. Tomlinson ◽  
Michael D. Sacco ◽  
Judy J. Chen ◽  
Anika N. Ali ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cell Size ◽  
Unknown Function ◽  
Suppressor Mutation ◽  
Gram Positive ◽  
Content Type ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

scholarly journals HAMLET, a Protein Complex from Human Milk, Has Bactericidal Activity and Enhances the Activity of Antibiotics against Pathogenic Streptococci

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Feiruz Alamiri ◽  
Kristian Riesbeck ◽  
Anders P. Hakansson
Keyword(s):  
Human Milk ◽  
Bactericidal Activity ◽  
Combination Treatment ◽  
Bacterial Species ◽  
Penicillin G ◽  
Resistant Bacteria ◽  
Tumoricidal Activity ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Transport Inhibitors

ABSTRACT HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex derived from human milk that was first described for its tumoricidal activity. Later studies showed that HAMLET also has direct bactericidal activity against select species of bacteria, with highest activity against Streptococcus pneumoniae. Additionally, HAMLET in combination with various antimicrobial agents can make a broad range of antibiotic-resistant bacterial species sensitive to antibiotics. Here, we show that HAMLET has direct antibacterial activity not only against pneumococci but also against Streptococcus pyogenes (group A streptococci [GAS]) and Streptococcus agalactiae (group B streptococci [GBS]). As with pneumococci, HAMLET treatment of GAS and GBS resulted in depolarization of the bacterial membrane, followed by membrane permeabilization and death, which was able to be inhibited by calcium and sodium transport inhibitors. Treatment of clinical antibiotic-resistant isolates of S. pneumoniae, GAS, and GBS with sublethal concentrations of HAMLET in combination with antibiotics decreased the MICs of the antibiotics into the sensitive range. This effect could also be blocked by ion transport inhibitors, suggesting that HAMLET’s bactericidal and combination treatment effects used similar mechanisms. Finally, we show that HAMLET potentiated the effects of erythromycin against erythromycin-resistant bacteria more effectively than penicillin G potentiated killing bacteria resistant to erythromycin. These results show that HAMLET effectively (i) kills three different species of pathogenic streptococci by similar mechanisms and also (ii) potentiates the activities of macrolides and lincosamides more effectively than combination treatment with beta-lactams. These findings suggest a potential therapeutic role for HAMLET in repurposing antibiotics currently causing treatment failures in patients.

scholarly journals Cohesion of Sister Chromosome Termini during the Early Stages of Sporulation in Bacillus subtilis

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Clare Willis ◽  
Jeff Errington ◽  
Ling Juan Wu
Keyword(s):  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Developmental Process ◽  
Time Lapse ◽  
Axial Filament ◽  
Content Type ◽  
Early Stages ◽  
Cell Compartments ◽  
Sister Chromosome

ABSTRACT During sporulation of Bacillus subtilis, the cell cycle is reorganized to generate separated prespore and mother cell compartments, each containing a single fully replicated chromosome. The process begins with reorganization of the nucleoid to form an elongated structure, the axial filament, in which the two chromosome origins are attached to opposite cell poles, with the remainder of the DNA stretched between these sites. When the cell then divides asymmetrically, the division septum closes around the chromosome destined for the smaller prespore, trapping the origin-proximal third of the chromosome in the prespore. A translocation pore is assembled through which a DNA transporter, SpoIIIE/FtsK, transfers the bulk of the chromosome to complete the segregation process. Although the mechanisms involved in attaching origin regions to the cell poles are quite well understood, little is known about other aspects of axial filament morphology. We have studied the behavior of the terminus region of the chromosome during sporulation using time-lapse imaging of wild-type and mutant cells. The results suggest that the elongated structure involves cohesion of the terminus regions of the sister chromosomes and that this cohesion is resolved when the termini reach the asymmetric septum or translocation pore. Possible mechanisms and roles of cohesion and resolution are discussed. IMPORTANCE Endospore formation in Firmicutes bacteria provides one of the most highly resistant life forms on earth. During the early stages of endospore formation, the cell cycle is reorganized so that exactly two fully replicated chromosomes are generated, before the cell divides asymmetrically to generate the prespore and mother cell compartments that are critical for the developmental process. Decades ago, it was discovered that just prior to asymmetrical division the two chromosomes enter an unusual elongated configuration called the axial filament. This paper provides new insights into the nature of the axial filament structure and suggests that cohesion of the normally separated sister chromosome termini plays an important role in axial filament formation.

scholarly journals Reclassification of the biocontrol agents Bacillus subtilis BY-2 and Tu-100 as Bacillus velezensis and insights into the genomic and specialized metabolite diversity of the species

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1121-1128 ◽  
Author(s):  
Alex J. Mullins ◽  
Yinshui Li ◽  
Lu Qin ◽  
Xiaojia Hu ◽  
Lihua Xie ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Natural Product ◽  
Type Species ◽  
Bacterial Species ◽  
Gene Clusters ◽  
Core Gene ◽  
Biocontrol Agents ◽  
Bacillus Velezensis ◽  
Content Type ◽  
Link Type

The genomes of two historical Bacillus species strains isolated from the roots of oilseed rape and used routinely in PR China as biocontrol agents to suppress Sclerotinia disease were sequenced. Average nucleotide identity (ANI) and digital DNA–DNA hybridization analyses demonstrated that they were originally misclassified as Bacillus subtilis and now belong to the bacterial species Bacillus velezensis . A broader ANI analysis of available Bacillus genomes identified 292 B. velezensis genomes that were then subjected to core gene analysis and phylogenomics. Prediction and dereplication of specialized metabolite biosynthetic gene clusters (BGCs) defined the prevalence of multiple antimicrobial-associated BGCs and highlighted the natural product potential of B. velezensis . By defining the core and accessory antimicrobial biosynthetic capacity of the species, we offer an in-depth understanding of B. velezensis natural product capacity to facilitate the selection and testing of B. velezensis strains for use as biological control agents.

scholarly journals A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
David M. Rothstein ◽  
David Lazinski ◽  
Marcia S. Osburne ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Rna Synthesis ◽  
Bacterial Species ◽  
Ethanol Stress ◽  
Temperature Sensitive ◽  
Content Type ◽  
Promoter Recognition ◽  
Bacillis Subtilis

ABSTRACT Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU, a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes.

scholarly journals Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

2015 ◽  
Vol 197 (14) ◽  
pp. 2374-2382 ◽  
Author(s):  
Samuel Million-Weaver ◽  
Ariana Nakta Samadpour ◽  
Houra Merrikh
Keyword(s):  
Bacillus Subtilis ◽  
Double Strand Breaks ◽  
Holliday Junction ◽  
Replication Forks ◽  
Dna Breaks ◽  
Site Specific ◽  
Content Type ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Replication Restart

ABSTRACTEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription inBacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.IMPORTANCEHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms forBacillus subtiliscells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

scholarly journals Watching DNA Replication Inhibitors in Action: Exploiting Time-Lapse Microfluidic Microscopy as a Tool for Target-Drug Interaction Studies in Mycobacterium

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Damian Trojanowski ◽  
Marta Kołodziej ◽  
Joanna Hołówka ◽  
Rolf Müller ◽  
Jolanta Zakrzewska-Czerwińska
Keyword(s):  
Nalidixic Acid ◽  
Bacterial Species ◽  
Time Lapse ◽  
Multidrug Resistant ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Target Drug ◽  
Causative Agents ◽  
Reporter Strains ◽  
Stalled Replication Forks

ABSTRACT Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria, which are the causative agents of severe diseases and which have profound impacts on global health. Here, we used a system of microfluidics, fluorescence microscopy, and target-tagged fluorescent reporter strains of Mycobacterium smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid, and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid and that nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, while griselimycin caused replisome instability, with the subsequent overinitiation of chromosome replication and overrelaxation of the nucleoid. In addition to study target-drug interactions, our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.).

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