Pathogens utilize a diverse set of signal transduction mechanisms to respond to host-derived stresses, with phosphotransfer-mediated two component systems (TCS) playing key roles in virulence factor regulation. Staphylococcus aureus encodes an alternative protease-mediated TCS prototype known as BlaRI involved in inducible β-lactam resistance. BlaR senses extracellular β-lactams, leading to activation of a cytoplasmic protease domain able to cleave DNA-bound BlaI dimers, de-repressing blaRI and blaZ (β-lactamase). The two known mycobacterial BlaRI-type systems in M. tuberculosis (Mtb) and M. abscessus ( Mab) are characterized by BlaR orthologs with conserved zinc metalloprotease domain but lacking an extracellular β-lactam binding domain. BlaIR Mtb and BlaIR Mab (renamed BlaIR to reflect inverted genomic organization) regulate β-lactamase expression ( Mtb only) and respiration (both Mtb and Mab). In this study, we have identified a second BlaRI-type system in Mab, MAB_4287-4288 (BlaIR2). Using RT-PCR and EMSA, we established that BlaIR2 Mab is encoded within a five gene operon, a unique characteristic in BlaRI-type systems, and is auto-regulatory. Identification of putative BlaI2 Mab binding motifs revealed a predicted regulon comprised of several genes involved in respiration, with some overlap with the BlaI1 Mab regulon. Finally, our data demonstrated that BlaIR2 Mab was also induced by the respiration inhibitor clofazimine (CFZ) similarly to BlaIR1 Mab, with evidence of possible crosstalk between BlaRI systems. Overall, this study established MAB_4287-4288 (BlaIR2 Mab) as a second BlaRI-type system in Mab, whose role may overlap or intersect that of BlaRI1 Mab. However, the activation mechanism and full role of BlaIR2 Mab in Mab stress responses and pathogenesis remains to be elucidated.

Competence is an important bioprocess for Streptococcus pneumoniae. Previously, we demonstrated that bacterial second messenger cyclic di-adenosine monophosphate (c-di-AMP) modulates pneumococcal competence. Surprisingly, a low c-di-AMP-producing strain, cdaA*, due to a mutation in the diadenylate cyclase CdaA, is highly susceptible to competence-stimulating peptide (CSP). In this study, we screened cdaA* suppressor mutations resistant to CSP to explore c-di-AMP signaling in S. pneumoniae. A total of 14 clones were sequenced, nine clones possessed mutations in the c-di-AMP phosphodiesterase Pde1, indicating that the susceptibility to CSP of cdaA* is correlated to c-di-AMP levels. Another two clones exhibited a mutation in FabT, a transcription factor controlling cell membrane fatty acid biosynthesis and metabolism. We further showed that deletion of fabT, disruption of the FabT-binding site within the P fabK promoter, deletion of a fabT activator BriC, or disruption of K + uptake in cdaA* all rescued the growth defect of cdaA* in media supplemented with CSP. Finally, we found that a c-di-AMP phosphodiesterase-null mutant with high levels of c-di-AMP is highly sensitive to treatment with either ethanol or Triton X-100, which could be corrected by reducing c-di-AMP levels through introducing point mutations in CdaA. Together, these findings indicate that c-di-AMP affects cell membrane integrity.

Acinetobacter baumannii is an opportunistic pathogen causing several infections that are increasingly difficult to treat due to its ability to rapidly gain antibiotic resistances. These resistances can arise due to mutations during the DNA damage response (DDR), through the activity of error-prone DNA polymerases, such as DNA polymerase V (DNA Pol V). Currently, the DDR in A. baumannii is not well understood and the regulation of genes encoding multiple copies of DNA Pol V is not fully characterized. Through genome wide mutagenesis, we have identified a novel TetR-like family regulator of genes that encode DNA Pol V, which we have named E rror- p rone p olymerase r egulator, EppR. We have found that EppR represses the expression of the genes encoding DNA Pol V and itself through direct binding to a conserved EppR motif in their promoters. Lastly, we show that EppR also regulates UmuDAb, previously identified as a regulator of genes encoding DNA Pol V. These two gene products are functionally required to ensure regulation of expression of genes encoding DNA Pol Vs and umuDAb. With these results, we propose a co-repressor model for the regulation of genes encoding DNA Pol V and umuDAb.

Queuosine (Q) is a modification of the wobble base in tRNAs that decode NAC codons. It is ubiquitous in bacteria, including many pathogens. Streptococcus mutans is an early colonizer of dental plaque biofilm and a key player in dental caries. Using a combination of genetic and physiological approaches, the predicted Q synthesis and salvage pathways were validated in this organism. These experiments confirmed that S. mutans can synthesize Q de novo through similar pathways found in Bacillus subtilis and Escherichia coli. However, S. mutans has a distinct salvage pathway compared to these model organisms, as it uses a transporter belonging to the Energy Coupling Factor (ECF) family controlled by a preQ 1-dependent riboswitch. Furthermore, Q levels in this oral pathogen depended heavily on the media composition, suggesting that micronutrients can affect Q-mediated translation efficiency.

Cyanobacteria developed oxygenic photosynthesis and represent the phylogenetic ancestors of chloroplasts. The model strain Anabaena sp. PCC 7120 grows as filaments of communicating cells and can form heterocysts, cells specialized for N 2 fixation. In the Anabaena genome, ORF all2390 is annotated as encoding a SulA homolog, but sequence similarity to SulA of model bacteria is unsignificant. We generated strains that lacked or overexpressed all2390, both of which showed instances of increased cell size, and observed that purified All2390 protein interfered with the in vitro polymerization of FtsZ. Heterocyst frequency diminished by all2390 inactivation and increased by all2390 overexpression. Overexpression retarded the dismantlement of Z-ring structures that sets commitment in the differentiating cells. Thus, All2390 can influence cell division affecting heterocyst differentiation. An All2390-GFP fusion protein localized to the thylakoidal membranes in vegetative cells and to the honeycomb membranes, which harbor photosynthetic complexes, in the heterocyst polar regions. Notably, all2390 expression strongly increased under high light, conditions under which the null mutant is unable to survive. Thus, All2390 appears essential for adaptation to high light conditions. We name All2390 ThyZ to reflect its dual role in FtsZ-ring dynamics and acclimation of thylakoidal membranes to increased light intensity.

In the human pathogen Staphylococcus aureus, branched-chain fatty acids (BCFAs) are the most abundant fatty acids in membrane phospholipids, and strains deficient for their synthesis experience BCFAs auxotrophy in laboratory culture and attenuated virulence during infection. Thus, membrane integrity is essential for S. aureus pathogenesis. Furthermore, the membrane of S. aureus is among the main targets for antibiotic therapy. Therefore, determining the mechanisms involved in BCFAs synthesis is critical to manage S. aureus infections. Here, we report that overexpression of the bona fide acyl-CoA synthetase gene mbcS (formerly SAUSA300_2542) restores BCFAs synthesis in strains lacking the canonical biosynthetic pathway catalyzed by the branched-chain a-keto acid dehydrogenase (BKDH) complex. We demonstrate that the acyl-CoA synthetase activity of MbcS activates branched-chain carboxylic acids, and is required by S. aureus to utilize the isoleucine derivative 2-methylbutyraldehyde to restore BCFAs synthesis in S. aureus. Based on the ability of some staphylococci to convert branched-chain aldehydes into their respective branched-chain carboxylic acids and our findings demonstrating that branched-chain aldehydes are in fact BCFAs precursors, we propose that MbcS promotes the scavenging of exogenous branched-chain carboxylic acids (BCCAs) and mediates branched-chain fatty acids synthesis via a de novo alternative pathway.

Urinary tract infection caused by Escherichia coli can lead to urosepsis, and the pathogens’ effectiveness in acquiring iron determines their virulence. The level of expression of the siderophore genes in different environments (M9 medium, artificial urine, and M9 supplemented with blood) was investigated using the CAS assay and qPCR. Aerobactin was found much more frequently in blood than in urine isolates (P=0.03), but the expression in artificial urine was 2x higher than in blood (P=0.03). We observed significant changes in the expression of entC, iro-2, iucA, and iroB bacterial genes between groups in artificial urine. The siderophores enterobactin, aerobactin and yersiniabactin (P=0.016), with the iha and hlyA genes, may represent markers of increased urosepsis risk. Moreover, proteomic profiles between groups were significantly different (ferritin-1, iron uptake system component EfeO, ferrous iron transport protein B, nitrate/nitrite response regulator protein NarL, protein HemY, ferrienterobactin receptor FepA, lipopolysaccharide export system protein LptA and 2Fe-2S ferredoxin). We also observed the positive association with urosepsis of IL-6, TNF, and CRP proteins between groups. In conclusion, risk factors for UTI-related sepsis may be related to the iron uptake system, and genetic and proteomic profiles may help in their identification.

The nosocomial bacterium Acinetobacter baumannii is protected from antibiotic treatment by acquiring antibiotic resistances and by forming biofilms. Cell attachment, one of the first steps in biofilm formation, is normally induced by environmental metabolites. We hypothesized that vanillic acid, the oxidized form of vanillin, a widely available metabolite with antimicrobial properties, may play a role in A. baumannii cell attachment. We first discovered that A. baumannii actively breaks down VA through the evolutionarily conserved vanABKP genes. These genes are under the control of the repressor VanR, which we show binds directly to VanR binding sites within the vanABKP genes bidirectional promoter. VA in turn counteracts VanR inhibition. We identified a VanR binding site and searched for it throughout the genome especially in pili encoding promoter genes. We found a VanR binding site in the pilus encoding csu operon promoter and showed that VanR binds specifically to it. As expected, a strain lacking VanR overproduces Csu pili and makes robust biofilms. Our study uncovers the role that VA plays in facilitating the attachment of A. baumannii cells to surfaces, a crucial step in biofilm formation. These findings provide valuable insights into a previously obscure catabolic pathway with significant clinical implications.

Bacterial pathogens possess the remarkable ability to sense and adapt to their constantly changing environments. For example, Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in aquatic settings and infects human hosts, employing dynamic strategies to ensure survival between diverse environments. In this study, we utilized RNA sequencing to investigate how V. cholerae responds to nitric oxide (NO), a common stressor encountered during infection. We found that cry1, encoding a photolyase, which repairs DNA damage caused by exposure to blue light—a stressor found in aquatic environments—was induced by NO. We observed that cry1 was activated upon exposure to blue light and Cry1 contributed to mitigating blue light-induced reactive oxygen species (ROS) production and stress responses. Cry1 is important for protecting cells against blue light-induced cell death. Furthermore, we showed that pre-induced Cry1 production, either by exposure to reactive nitrogen species (RNS) in vitro or in mice, enhanced V. cholerae’s resistance to blue light. Additionally, we found that V. cholerae Cry1 and the E. coli ortholog PhrB were crucial in resisting reactive oxygen species (ROS). Moreover, cry1 expression was regulated by RpoE and the anti-sigma factor ChrR, with two cysteine residues in ChrR playing vital roles in sensing RNS and blue light-induced ROS, thereby modulating cry1 expression. Collectively, our findings suggest that V. cholerae encounters host-derived NO during infection, and the subsequent induction of Cry1 primes the bacterium effectively for challenges in aquatic environments abundant in blue light once it exits the host gut.

Microbial communities are gaining ground in biotechnology, as they offer many advantages over single-organism monocultures. To make microbial communities competitive as a biotechnological platform, it is essential that we develop strategies to engineering and optimizing their functionality. To this end, most efforts have focused on genetic manipulations. An alternative and also very promising strategy is to optimize the function of microbial communities by rationally engineering their environment and culture conditions. A major challenge is that the combinatorial space of environmental factors is enormous. Furthermore, environmental factors such as temperature, pH, nutrient composition, etc., generally combine their effects in complex, non-additive ways. In this piece, we overview the origins and consequences of these “interactions” between environmental factors, and discuss how they have been built into statistical models of microbial community function to identify optimal environmental conditions. We also overview alternative “top-down” approaches, such as genetic algorithms, to finding combinations of environmental factors that optimize the function of microbial consortia. By providing a brief summary of the state of this field, we hope to stimulate the development of novel methodologies to rationally manipulate and optimize microbial communities through their environment.

Lysine acetylation is an evolutionarily conserved protein modification that regulates different cellular pathways. The acetyl moiety can be transferred to the lysine side chain in two ways, enzymatically by lysine acetyltransferases and non-enzymatic. Usually, acetyl coenzyme A is the donor agent, although acetyl phosphate is the main regulator of acetylation in bacteria. The removal of the acetyl group occurs exclusively enzymatically. In prokaryotes, lysine acetyltransferases are grouped by a core structural domain architecture in the protein superfamily Gcn5-related N-acetyltransferase (GNAT). With different implications, these enzymes can acetylate the amino group of small molecules, metabolites, peptides, and proteins. As well as mitochondria, chemical acetylation has also been demonstrated as a global regulatory mechanism in bacteria. This review presents current knowledge of acetylation mechanisms and functional implications in bacteria metabolism. Additionally, the advances in mass spectrometry for studying this PTM, including relative quantification and stoichiometry quantification, and how these methods have allowed researchers to elucidate the biological significance in bacteria are reviewed.

Since its inception in the 1930’s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of < 100 nm thick and with parasites being larger than 1 µm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate and speed-up the structural characterisation of biological samples, offering new imaging modalities for parasitology. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 µm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of the STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of parasites.

Shigella flexneri type III effector OspC3 was recently found to evade host pyroptosis by targeting caspase-11 for ADP-riboxanation. The ADP-riboxanase activity was shared by its paralogues, OspC1 and OspC2, whereas the host substrates of them are still unknown. To solve this problem, we employed eAf1521 enrichment coupled with mass spectrometry (MS)-based proteomics to profile the host substrates of OspC1. In this study, we identified HERC5 as a host target of OspC1. As described previously, HERC5 functions as an E3 ISG15 ligase and catalyzes the ISGylation of a huge subset of host and pathogen proteins upon bacterial infection. Here, we show that S. flexneri hijacks host ISGylation pathway by OspC1-catalyzed ADP-riboxanation of HERC5 to promote its own survival and proliferation in host cells.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here we use a combination of genetics, quantitative phenotyping, and gene expression assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆ trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name Trm B su ppressor, or TbsP. TbsP is required for adhesion to abiotic surfaces (i.e. biofilm formation), and maintains wild type cell morphology, and motility. TrmB and TbsP jointly regulate the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP co-regulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Its success as a pathogen is dependent on an arsenal of secreted virulence factors which target host cell organelles. In this report, we have characterized the effects of two proteins known to be secreted by Mtb on eukaryotic mitochondria: ESAT-6 and a member of the poorly-described PE/PPE protein family, PE17. Intracellular expression of ESAT-6 in A549 cells did not induce mitochondrial fragmentation. In contrast, expression of PE17 caused extensive mitochondrial fragmentation and an overall reduction in mass. Further characterization of the effects of PE17 within host cells revealed a dramatic decrease in the mass of the trans-Golgi network and the Golgi stack with lesser but significant decreases in late endosomes and lysosomes. The endoplasmic reticulum and organelles of the early endocytic pathway were not significantly affected. PE17 specifically colocalized with large, cytoplasmic lipid droplets via a C-terminal domain, suggesting a significant role for PE17 in the disruption of multiple host cell organelles and a novel interaction with host lipid droplets.

Mycobacterium tuberculosis has co-evolved with its host to evade and modulate host cellular processes. Few PE/PPE proteins of Mycobacterium have been reported to modulate cell death pathways and determine the infection outcome. This study investigates role of one such uncharacterized protein PE_PGRS45/Rv2615c in host cell death and immuno-modulation. In-silico analysis predicted similarity of Rv2615c with APAF1-apoptosome and involvement in Cysteine-type endo-peptidase activation during apoptosis. In-vitro experiments with THP-1 macrophages confirmed the Caspase-dependent apoptosis inducing potential of Rv2615c. Several PE_PGRS proteins have been reported to be TLR-agonist. Docking showed preferential binding of Rv2615c with TLR4 than TLR2. Up-regulation of TLR4-HLA-DR-MyD88-NF-ƙB-TNF‐α in Rv2615c-stimulated THP-1 macrophages was observed. To investigate the mechanism underpinning the apoptotic function, a thorough sequence scan of Rv2615c revealed the presence of eukaryotic CARD-like domain in it. In-silico studies showed binding affinity of Rv2615c’s CARD-like domain with APAF1-CARD and Caspase9-CARD, attributing Rv2615c’s role in apoptosis. Since, Rv2615c is reported to be upregulated in dormancy which suggest that it may be one of the unexplored effectors employed by Mtb for its persistence via silent cell-to-cell spread of infection by inducing macrophage apoptosis. Additionally, Rv2615c’s interaction with TLR4 implicates its engagement in host-pathogen interaction.