Arginine methylation sites on SepIVA help balance elongation and septation in Mycobacterium smegmatis.

The growth of mycobacterial cells requires successful coordination between elongation and septation. However, it is not clear which factors mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of its methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG and that this regulation may impact polar elongation. Finally, we map SepIVA's two regulatory functions to different ends of the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

Mycobacterial serine/threonine phosphatase PstP is phosphoregulated and localized to mediate control of cell wall metabolism.

The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven serine/threonine protein kinases phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated, but the role of its phosphorylation in regulating its activity has been unclear. In this study, we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two regulators of peptidoglycan in Mtb, FhaA, and Wag31. We also show that a phosphomimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulators FhaA, Wag31, CwlM, PknB, and PknA, and that the corresponding mutation in Mycobacterium smegmatis causes misregulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.

Localized Production of Cell Wall Precursors May Be Critical for Regulating the Mycobacterial Cell Wall.

The paper "Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a nonredundant role for RodA in mycobacteria" by Melzer et al. (E. S. Melzer, T. Kado, A. Garcia-Heredia, K. R. Gupta, et al., J Bacteriol 204:e00540-21, 2022, https://doi.org/10.1128/JB.00540-21) presents several new observations about the localization and function of cell wall enzymes in Mycobacterium smegmatis and their responses to stress. This work illustrates some important aspects of cell wall physiology in mycobacteria and also points to a new model for how peptidoglycan synthesis may be organized in pole-growing bacteria.

In Mycobacterium abscessus, the Stringent Factor Rel Regulates Metabolism but Is Not the Only (p)ppGpp Synthase.

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the nontuberculous mycobacterial pathogen, Mycobacterium abscessus (Mab). We found that deletion of rel does not ablate (p)ppGpp synthesis and that rel does not provide a survival advantage in several stress conditions or in antibiotic treatment. Transcriptional data show that RelMab is involved in regulating expression of anabolism and growth genes in the stationary phase. However, it does not activate transcription of stress response or antibiotic resistance genes and actually represses transcription of many antibiotic resistance genes. This work shows that there is an unannotated (p)ppGpp synthetase in Mab. IMPORTANCE In this study, we examined the functional roles of the stringent factor Rel in Mycobacterium abscessus (Mab). In most species, stringent factors synthesize the alarmone (p)ppGpp, which globally alters transcription to promote growth arrest and survival under stress and in antibiotic treatment. Our work shows that in Mab, an emerging pathogen that is resistant to many antibiotics, the stringent factor Rel is not solely responsible for synthesizing (p)ppGpp. We find that RelMab downregulates many metabolic genes under stress but does not upregulate stress response genes and does not promote antibiotic tolerance. This study implies that there is another critical but unannotated (p)ppGpp synthetase in Mab and suggests that RelMab inhibitors are unlikely to sensitize Mab infections to antibiotic treatment.

Phosphorylation on PstP Regulates Cell Wall Metabolism and Antibiotic Tolerance in Mycobacterium smegmatis.

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the serine/threonine phosphatase PstP, in the model organism Mycobacterium smegmatis We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phosphomimetic mutation, pstP T171E, slows growth, misregulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.IMPORTANCE Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

Mycobacterium abscessus Cells Have Altered Antibiotic Tolerance and Surface Glycolipids in Artificial Cystic Fibrosis Sputum Medium.

Mycobacterium abscessus is a biofilm-forming, multidrug-resistant nontuberculous mycobacterial (NTM) pathogen increasingly found in cystic fibrosis patients. Antibiotic treatment for these infections is often unsuccessful, partly due to M. abscessus's high intrinsic antibiotic resistance. It is not clear whether antibiotic tolerance caused by biofilm formation also contributes to poor treatment outcomes. We studied the surface glycolipids and antibiotic tolerance of M. abscessus biofilms grown in artificial cystic fibrosis sputum (ACFS) medium to determine how they are affected by nutrient conditions that mimic infection. We found that M. abscessus displays more of the virulence lipid trehalose dimycolate when grown in ACFS than when grown in standard lab medium. In ACFS medium, biofilm-associated cells were more antibiotic tolerant than planktonic cells in the same well. This contrasts with standard lab media, where both biofilm and planktonic cells are highly antibiotic tolerant. These results indicate that M. abscessus cell physiology in biofilms depends on environmental factors and that nutrient conditions found within cystic fibrosis infections could contribute to both increased virulence and antibiotic tolerance.

Characterization of Conserved and Novel Septal Factors in Mycobacterium smegmatis.

Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria.

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1's catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress.

MmpL3, Wag31 and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis.

Mycobacteria expand their cell walls at the cell poles in a manner that is not well described at the molecular level. In this study, we identify a new polar factor, PlrA, that is involved in restricting peptidoglycan metabolism to the cell poles in Mycobacterium smegmatis. We establish that only the N-terminal membrane domain of PlrA is essential. We show that depletion of plrA pheno-copies depletion of polar growth factor Wag31, and that PlrA is involved in regulating the Wag31 polar foci.

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus.

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.

The complex logic of stringent response regulation in Caulobacter crescentus: starvation signalling in an oligotrophic environment.

Bacteria rapidly adapt to nutritional changes via the stringent response, which entails starvation-induced synthesis of the small molecule, ppGpp, by RelA/SpoT homologue (Rsh) enzymes. Binding of ppGpp to RNA polymerase modulates the transcription of hundreds of genes and remodels the physiology of the cell. Studies of the stringent response have primarily focused on copiotrophic bacteria such as Escherichia coli; little is known about how stringent signalling is regulated in species that live in consistently nutrient-limited (i.e. oligotrophic) environments. Here we define the input logic and transcriptional output of the stringent response in the oligotroph, Caulobacter crescentus. The sole Rsh protein, SpoT(CC), binds to and is regulated by the ribosome, and exhibits AND-type control logic in which amino acid starvation is a necessary but insufficient signal for activation of ppGpp synthesis. While both glucose and ammonium starvation upregulate the synthesis of ppGpp, SpoT(CC) detects these starvation signals by two independent mechanisms. Although the logic of stringent response control in C. crescentus differs from E. coli, the global transcriptional effects of elevated ppGpp are similar, with the exception of 16S rRNA transcription, which is controlled independently of spoT(CC). This study highlights how the regulatory logic controlling the stringent response may be adapted to the nutritional niche of a bacterial species.

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum.

Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole or how it is organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. In this study we used alanine scanning mutagenesis to identify Wag31 residues involved in cell morphogenesis. Our data show that Wag31 helps to control proper septation as well as new and old pole elongation. We have identified key amino acid residues involved in these essential functions. Enzyme assays revealed that Wag31 interacts with lipid metabolism by modulating acyl-CoA carboxylase (ACCase) activity. We show that Wag31 does not control polar growth by regulating the localization of cell wall precursor enzymes to the Intracellular Membrane Domain, and we also demonstrate that phosphorylation of Wag31 does not substantively regulate peptidoglycan metabolism. This work establishes new regulatory functions of Wag31 in the mycobacterial cell cycle and clarifies the need for new molecular models of Wag31 function.

Peptidoglycan Recycling Promotes Outer Membrane Integrity and Carbapenem Tolerance in Acinetobacter baumannii.

ß-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. Carbapenem ß-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections, but treatment failure is increasingly prevalent. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate tolerance to high-level meropenem treatment, form spheroplasts upon exposure to the antibiotic, and revert to normal growth after antibiotic removal. Using transcriptomics and genetic screens, we show that several genes associated with outer membrane integrity maintenance and efflux promote tolerance, likely by limiting entry into the periplasm. Genes associated with peptidoglycan homeostasis in the periplasm and cytoplasm also answered our screen, and their disruption compromised cell envelope barrier function. Finally, we defined the enzymatic activity of the tolerance determinants penicillin-binding protein 7 (PBP7) and ElsL (a cytoplasmic ld-carboxypeptidase). These data show that outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to A. baumannii meropenem tolerance. IMPORTANCE Carbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting the urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote the acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well understood. Here, we characterized the role of peptidoglycan recycling in outer membrane integrity maintenance and meropenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlight physiological processes that could be targeted to improve antimicrobial treatment.

Genetic and computational identification of a conserved bacterial metabolic module.

We have experimentally and computationally defined a set of genes that form a conserved metabolic module in the alpha-proteobacterium Caulobacter crescentus and used this module to illustrate a schema for the propagation of pathway-level annotation across bacterial genera. Applying comprehensive forward and reverse genetic methods and genome-wide transcriptional analysis, we (1) confirmed the presence of genes involved in catabolism of the abundant environmental sugar myo-inositol, (2) defined an operon encoding an ABC-family myo-inositol transmembrane transporter, and (3) identified a novel myo-inositol regulator protein and cis-acting regulatory motif that control expression of genes in this metabolic module. Despite being encoded from non-contiguous loci on the C. crescentus chromosome, these myo-inositol catabolic enzymes and transporter proteins form a tightly linked functional group in a computationally inferred network of protein associations. Primary sequence comparison was not sufficient to confidently extend annotation of all components of this novel metabolic module to related bacterial genera. Consequently, we implemented the Graemlin multiple-network alignment algorithm to generate cross-species predictions of genes involved in myo-inositol transport and catabolism in other alpha-proteobacteria. Although the chromosomal organization of genes in this functional module varied between species, the upstream regions of genes in this aligned network were enriched for the same palindromic cis-regulatory motif identified experimentally in C. crescentus. Transposon disruption of the operon encoding the computationally predicted ABC myo-inositol transporter of Sinorhizobium meliloti abolished growth on myo-inositol as the sole carbon source, confirming our cross-genera functional prediction. Thus, we have defined regulatory, transport, and catabolic genes and a cis-acting regulatory sequence that form a conserved module required for myo-inositol metabolism in select alpha-proteobacteria. Moreover, this study describes a forward validation of gene-network alignment, and illustrates a strategy for reliably transferring pathway-level annotation across bacterial species.

Septal Class A Penicillin-Binding Protein Activity and ld-Transpeptidases Mediate Selection of Colistin-Resistant Lipooligosaccharide-Deficient Acinetobacter baumannii.

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS- colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS-A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS-A. baumannii PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS-A. baumannii PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS-A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS-A. baumanniiIMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS-Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic ß-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS-A. baumannii.

The mycobacterial cell envelope - a moving target.

Mycobacterium tuberculosis, the leading cause of death due to infection, has a dynamic and immunomodulatory cell envelope. The cell envelope structurally and functionally varies across the length of the cell and during the infection process. This variability allows the bacterium to manipulate the human immune system, tolerate antibiotic treatment and adapt to the variable host environment. Much of what we know about the mycobacterial cell envelope has been gleaned from model actinobacterial species, or model conditions such as growth in vitro, in macrophages and in the mouse. In this Review, we combine data from different experimental systems to build a model of the dynamics of the mycobacterial cell envelope across space and time. We describe the regulatory pathways that control metabolism of the cell wall and surface lipids in M. tuberculosis during growth and stasis, and speculate about how this regulation might affect antibiotic susceptibility and interactions with the immune system.

Mycobacterium smegmatis HtrA Blocks the Toxic Activity of a Putative Cell Wall Amidase.

Mycobacterium tuberculosis, the causative agent of tuberculosis, withstands diverse environmental stresses in the host. The periplasmic protease HtrA is required only to survive extreme conditions in most bacteria but is predicted to be essential for normal growth in mycobacteria. We confirm that HtrA is indeed essential in Mycobacterium smegmatis and interacts with another essential protein of unknown function, LppZ. However, the loss of any of three unlinked genes, including those encoding Ami3, a peptidoglycan muramidase, and Pmt, a mannosyltransferase, suppresses the essentiality of both HtrA and LppZ, indicating the functional relevance of these genes' protein products. Our data indicate that HtrA-LppZ is required to counteract the accumulation of active Ami3, which is toxic under the stabilizing influence of Pmt-based mannosylation. This suggests that HtrA-LppZ blocks the toxicity of a cell wall enzyme to maintain mycobacterial homeostasis.

Two-component signaling systems and cell cycle control in Caulobacter crescentus.

Recent work on the regulation ofprokaryotic growth and development by two-component systems (TCS) has revealed unsuspected levels of complexity. In the dimorphic freshwater bacterium Caulobacter crescentus, TCS provide stringent temporal and spatial control of cellular development and cell-cycle progression. While the environmental signals modulating TCS regulatory networks are largely unknown, the components of the network and their interactions with each other are increasingly well-defined. Here, we present an overview of TCS regulation of cell-cycle control in C. crescentus.

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.

For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE: Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.