Molecular Microbiology最新文献

Many bacteria possess proteasomes and a tagging system that is functionally analogous to the ubiquitin system. In this system, Pup, the tagging protein, marks protein targets for proteasomal degradation. Despite the analogy to the ubiquitin system, where the ubiquitin tag is recycled, it remained unclear whether Pup is similarly recycled, given how the bacterial proteasome does not include a depupylase. We previously showed in vitro that as Pup lacks effective proteasome degradation sites, it is released from the proteasome following target degradation, remaining conjugated to a degradation fragment that can be later depupylated. Here, we tested this model in Mycobacterium smegmatis, using a Pup mutant that is effectively degraded by the proteasome. Our findings indicate that Pup recycling not only occurs in vivo but is also essential to maintain normal pupylome levels and to support bacterial survival under starvation conditions. Accordingly, Pup recycling is an essential process in the mycobacterial Pup-proteasome system.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab's β-lactamase (Bla_Mab) that mediates β-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the β-lactamase BlaZ. BlaR binds extracellular β-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mycobacterium tuberculosis (Mtb) encodes homologs of BlaRI (which we will denote as BlaIR to reflect the inverted gene order in mycobacteria) that regulate not only the Mtb β-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaIR_Mtb in Mab and hypothesized that they regulate bla_Mab. Surprisingly, neither deletion of blaIR_Mab nor overexpression of only blaI_Mab altered bla_Mab expression or β-lactam susceptibility. However, BlaI_Mab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaI_Mtb. Prompted by evidence that respiration inhibitors including clofazimine induce the BlaI regulon in Mtb, we found that clofazimine triggers induction of blaIR_Mab and its downstream regulon. Highlighting an important role for BlaIR_Mab in adapting to disruptions in energy metabolism, constitutive repression of the BlaI_Mab regulon rendered Mab highly susceptible to clofazimine. In addition to our unexpected findings that BlaIR_Mab does not regulate β-lactam resistance, this study highlights the novel role of mycobacterial BlaRI-type regulators in regulating electron transport and respiration.

FliL is a bacterial flagellar protein demonstrated to associate with, and regulate ion flow through, the stator complex in a diverse array of bacterial species. FliL is also implicated in additional functions such as stabilizing the flagellar rod, modulating rotor bias, sensing the surface, and regulating gene expression. How can one protein do so many things? Its location is paramount to understanding its numerous functions. This review will look at the evidence, attempt to resolve some conflicting findings, and offer new thoughts on FliL.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

DNA viruses recognize viral DNA and package it into virions. Specific recognition is needed to distinguish viral DNA from host cell DNA. The λ-like Escherichia coli phages are interesting and good models to examine genome packaging by large DNA viruses. Gifsy-1 is a λ-like Salmonella phage. Gifsy-1's DNA packaging specificity was compared with those of closely related phages λ, 21, and N15. In vivo packaging studies showed that a Gifsy-1-specific phage packaged λ DNA at ca. 50% efficiency and λ packages Gifsy-1-specific DNA at ~30% efficiency. The results indicate that Gifsy-1 and λ share the same DNA packaging specificity. N15 is also shown to package Gifsy-1 DNA. Phage 21 fails to package λ, N15, and Gifsy-1-specific DNAs; the efficiencies are 0.01%, 0.01%, and 1%, respectively. A known incompatibility between the 21 helix-turn-helix motif and cosB^λ is proposed to account for the inability of 21 to package Gifsy-1 DNA. A model is proposed to explain the 100-fold difference in packaging efficiency between λ and Gifsy-1-specific DNAs by phage 21. Database sequences of enteric prophages indicate that phages with Gifsy-1's DNA packaging determinants are confined to Salmonella species. Similarly, prophages with λ DNA packaging specificity are rarely found in Salmonella. It is proposed that λ and Gifsy-1 have diverged from a common ancestor phage, and that the differences may reflect adaptation of their packaging systems to host cell differences.

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.