In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2-Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Lastly, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

In E. coli K-12, absence of dephospho-PtsN, is thought to cause an L-leucine sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val) sensitive acetohyrodxyacid synthase I (AHAS I), that renders residual AHAS activity susceptible to inhibition by Leu and K+.  This is thought to lead to AHAS insufficiency and a requirement for isoleucine (Ile). Herein we provide an alternate mechanism for LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS, indicate that impaired expression of the ilvBN operon jointly caused by the absence of dephospho-PtsN and the presence of Leu coupled to Leu mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α-ketobutyrate level, mediate elevation of ilvBN expression, and alleviate the LeuS. The requirement of dephospho-PtsN as a positive regulator of ilvBN expression, may buffer Ile biosynthesis against Leu mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realised in certain environments that impair AHAS function.

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.