Transposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions.

Mass transit environments, specifically, urban subways, are distinct microbial environments with high occupant densities, diversities, and turnovers, and they are thus especially relevant to public health. Despite this, only three culture-independent subway studies have been performed, all since 2013 and all with widely differing designs and conclusions. In this study, we profiled the Boston subway system, which provides 238 million trips per year overseen by the Massachusetts Bay Transportation Authority (MBTA). This yielded the first high-precision microbial survey of a variety of surfaces, ridership environments, and microbiological functions (including tests for potential pathogenicity) in a mass transit environment. Characterizing microbial profiles for multiple transit systems will become increasingly important for biosurveillance of antibiotic resistance genes or pathogens, which can be early indicators for outbreak or sanitation events. Understanding how human contact, materials, and the environment affect microbial profiles may eventually allow us to rationally design public spaces to sustain our health in the presence of microbial reservoirs.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

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 , 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 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. 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 , but other proteins have critical regulatory functions that may be unique to the actinomycetes.