Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein\'s central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations\' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

BACKGROUND: Bacterial RNA polymerase holoenzyme requires sigma70 factors to start transcription by identifying promoter elements. Cyanobacteria possess multiple sigma70 factors to adapt to a wide variety of ecological niches. These factors are grouped into two categories: primary sigma factor initiates transcription of housekeeping genes during normal growth conditions, while alternative sigma factors initiate transcription of specific genes under particular conditions. However, the present classification does not consider the modular organization of their structural domains, introducing therefore multiple functional and structural biases. A comprehensive analysis of this protein family in cyanobacteria is needed to address these limitations.
RESULTS: We investigated the structure and evolution of sigma70 factors in cyanobacteria, analyzing their modular architecture and variation among unicellular, filamentous, and heterocyst-forming morphotypes. 4,193 sigma70 homologs were found with 59 distinct modular patterns, including six essential and 29 accessory domains, such as DUF6596. 90% of cyanobacteria typically have 5 to 17 sigma70 homologs and this number likely depends on the strain morphotype, the taxonomic order and the genome size. We classified sigma70 factors into 12 clans and 36 families. According to taxonomic orders and phenotypic traits, the number of homologs within the 14 main families was variable, with the A.1 family including the primary sigma factor since this family was found in all cyanobacterial species. The A.1, A.5, C.1, E.1, J.1, and K.1 families were found to be key sigma families that distinguish heterocyst-forming strains. To explain the diversification and evolution of sigma70, we propose an evolutionary scenario rooted in the diversification of a common ancestor of the A1 family. This scenario is characterized by evolutionary events including domain losses, gains, insertions, and modifications. The high occurrence of the DUF6596 domain in bacterial sigma70 proteins, and its association with the highest prevalence observed in Actinobacteria, suggests that this domain might be important for sigma70 function. It also implies that the domain could have emerged in Actinobacteria and been transferred through horizontal gene transfer.
CONCLUSIONS: Our analysis provides detailed insights into the modular domain architecture of sigma70, introducing a novel robust classification. It also proposes an evolutionary scenario explaining their diversity across different taxonomical orders.

Promoter sequences are important genetic control elements. Through their interaction with RNA polymerase they determine transcription strength and specificity, thereby regulating the first step in gene expression. Consequently, they can be targeted as elements to control predictability and tuneability of a genetic circuit, which is essential in applications such as the development of robust microbial cell factories. This review considers the promoter elements implicated in the three stages of transcription initiation, detailing the complex interplay of sequence-specific interactions that are involved, and highlighting that DNA sequence features beyond the core promoter elements work in a combinatorial manner to determine transcriptional strength. In particular, we emphasize that, aside from promoter recognition, transcription initiation is also defined by the kinetics of open complex formation and promoter escape, which are also known to be highly sequence specific. Significantly, we focus on how insights into these interactions can be manipulated to lay the foundation for a more rational approach to promoter engineering.

Promoter-specific activation of transcript initiation provides an important regulatory device in Escherichia coli and Salmonella. Here, we describe the different mechanisms that operate, focusing on how they have evolved to manage the \"housekeeping\" bacterial transcription machinery. Some mechanisms involve assisting the bacterial DNA-dependent RNA polymerase or replacing or remodeling one of its subunits. Others are directed to chromosomal DNA, improving promoter function, or relieving repression. We discuss how different activators work together at promoters and how the present complex network of transcription factors evolved.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism\'s survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

The Gram-positive model organism Bacillus subtilis responds to environmental stressors by activating the alternative sigma factor σB. The sensing apparatus upstream of σB activation is thought to consist of cytoplasmic stressosomes-megadalton-sized protein complexes that include five paralogous proteins known as RsbRs. The RsbRs are presumed to be involved in stress sensing and the subsequent response. Perturbations to the RsbR complement in stressosomes by engineering cells that produce only one of the RsbR paralogs (\"single-RsbR strains\") lead to altered σB response dynamics with respect to timing and magnitude. Here, we asked whether such changes to σB response dynamics impact the relative fitness of a strain. We competed strain pairs with different RsbR complements under ethanol and sodium chloride stress and found not only differences in relative fitness among wild-type and single-RsbR strains but also different relative fitness values in the two different stressors. We found that the presence of RsbRA, which dominates the wild-type σB response, enhances fitness in ethanol but is detrimental to fitness in NaCl. Meanwhile, RsbRD-only cells were among the most fit in NaCl. Strains producing hybrid RsbR fusion proteins displayed different fitness values that depended on the RsbR proteins from which they were derived. Our results here suggest that σB response dynamics can impact fitness, highlighting the physiological importance of the unusual stressosome-based general stress response system of B. subtilis.
OBJECTIVE: The model bacterium Bacillus subtilis uses cytoplasmic multiprotein complexes, termed stressosomes, to activate the alternative sigma factor σB when facing environmental stresses. We have previously shown that genetically manipulating the complement of putative sensor proteins in stressosomes can alter the dynamics of the σB response in terms of its magnitude and timing. However, it is unknown whether these response dynamics impact the fitness of cells challenged by environmental stressors. Here, we examine the fitness of strains with different σB responses by competing strain pairs in exponential-phase co-cultures under environmental stress. We find that strains with different response dynamics show different competitive indices that differ by stressor. These results suggest that the dynamics of the σB response can affect the fitness of cells facing environmental stress, highlighting the relevance of different σB dynamics.

In Escherichia coli, one of the best understood microorganisms, much can still be learned about the basic interactions between transcription factors and promoters. When a cAMP-deficient cya mutant is supplied with maltose as the main carbon source, mutations develop upstream from the two genes malT and sdaC. Here, we explore the regulation of the two promoters, using fluorescence-based genetic reporters in combination with both spontaneously evolved and systematically engineered cis-acting mutations. We show that in the cya mutant, regulation of malT and sdaC evolves toward cAMP-independence and increased expression in the stationary phase. Furthermore, we show that the location of the cAMP receptor protein (Crp) binding site upstream of malT is important for alternative sigma factor usage. This provides new insights into the architecture of bacterial promoters and the global interplay between Crp and sigma factors in different growth phases.IMPORTANCEThis work provides new general insights into (1) the architecture of bacterial promoters, (2) the importance of the location of Class I Crp-dependent promoters, and (3) the global interplay between Crp and sigma factors in different growth phases.

Vibrio species are motile gram-negative bacteria commonly found in aquatic environments. Vibrio species include pathogenic as well as non-pathogenic strains. Pathogenic Vibrio species have been reported in invertebrates and humans, whereas non-pathogenic strains are involved in symbiotic relationships with their eukaryotic hosts. These bacteria are also able to adapt to fluctuations in temperature, salinity, and pH, in addition to oxidative stress, and osmotic pressure in aquatic ecosystems. Moreover, they have also developed protective mechanisms against the immune systems of their hosts. Vibrio species accomplish adaptation to changing environments outside or inside the host by altering their gene expression profiles. To this end, several sigma factors specifically regulate gene expression, particularly under stressful environmental conditions. Moreover, other sigma factors are associated with biofilm formation and virulence as well. This review discusses different types of sigma and anti-sigma factors of Vibrio species involved in virulence and regulation of gene expression upon changes in environmental conditions. The evolutionary relationships between sigma factors with various physiological roles in Vibrio species are also discussed extensively.

A rogue, plasmid-encoded sigma factor that kills Bacillus subtilis is the focus of a new study by A. T. Burton, D. Pospíšilová, P. Sudzinová, E. V. Snider, A. M. Burrage, L. Krásný, and D. B. Kearns (J Bacteriol 205:e00112-23, 2023, https://doi.org/10.1128/jb.00112-23). The authors demonstrate that SigN is toxic in its own right, causing cell death by potently outcompeting the housekeeping sigma factor for access to RNA polymerase.

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ54-interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter.