Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio‐temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high‐throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini‐colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus . By tracking filamentous cells, we show that the chromosomal origin at the old‐pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position‐dependent silencing is regulated by division.
Quantitative spatial distributions of ribosomes (S2‐YFP) and RNA polymerase (RNAP; β′‐yGFP) in live Escherichia coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, nucleoid–ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55 000 ribosomes. Only 10–15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome‐rich endcaps. The predominant observed diffusion coefficient of ribosomes is D ribo = 0.04 µm 2 s −1 , attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of subdiffusion, as would arise from tethering of ribosomes to the DNA. The degree of DNA–ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome‐rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force.
Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non‐pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli , enteropathogenic E. coli , enteroinvasive E. coli , K1 and Shigella ] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long‐term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.
With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills. Oufti is an interactive, open source software package built for high throughput quantification of microscopy images. Oufti performs cell detection, localization of diffraction‐limited spots as well as boundary determination of image objects beyond the diffraction limit. Due to parallel processing, it is able to perform these tasks on large (>1Gb) datasets, which can be further expanded by high performance computing centers. Oufti also provides post‐processing tools for the statistical characterization of data following image analysis.
High levels of the intracellular signalling molecule cyclic diguanylate (c‐di‐GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c‐di‐GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c‐di‐GMP‐responsive transcriptional regulator in Pseudomonas aeruginosa . FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c‐di‐GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c‐di‐GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c‐di‐GMP. FleQ binds radiolabelled c‐di‐GMP in vitro . FleQ does not have amino acid motifs that resemble previously defined c‐di‐GMP binding domains. Our results show that FleQ is a new type of c‐di‐GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa .
P>Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.
Vibrio cholerae codes for 13 toxin–antitoxin (TA) loci all located within the superintegron on chromosome II. We show here that the two higBA TA loci of V. cholerae encode functional toxins, HigB‐1 and HigB‐2, whose ectopic expression inhibits cell growth of Escherichia coli , and functional antitoxins, HigA‐1 and HigA‐2, which counteract the toxicity of the cognate toxins. Three hours of ectopic expression of the HigB toxins resulted in bacteriostasis without any detectable loss of cell viability. The HigB toxins inhibited translation by cleavage of mRNA. Efficient mRNA cleavage occurred preferentially within the translated part of a model mRNA and only when the mRNA was translatable. Promoter analysis in V. cholerae and E. coli showed that the two higBA loci are both transcribed into bi‐cistronic mRNAs and that the higBA ‐2 mRNA is leaderless. Transcription of the two higBA loci was strongly induced by amino acid (aa) starvation in V. cholerae and E. coli , indicating that the regulatory mechanisms of transcriptional induction are conserved across the two species. Both higBA loci stabilized a test‐plasmid very efficiently in E. coli , raising the possibility that the loci contribute to maintain genetic stability of the V. cholerae superintegron. Based on these results we discuss the possible biological functions of the TA loci of V. cholerae .
We have developed a system for the simultaneous labelling of two specific chromosomal sites using two different fluorescent ParB/ parS systems. Using this, we demonstrate that the two chromosome arms are spatially arranged in newborn cells such that markers on the left arm of the chromosome lie in one half of the cell and markers on the right arm of the chromosome lie in the opposite half. This is achieved by reorganizing the chromosome arms of the two nucleoids in pre‐division cells relative to the cell quarters. The spatial reorganization of the chromosome arms ensures that the two replication forks remain in opposite halves of the cell during replication. The relative orientation of the two reorganized nucleoids in pre‐division cells is not random. Approximately 80% of dividing cells have their nucleoids oriented in a tandem configuration.
In E scherichia coli , acetylation of proteins at lysines depends largely on a non‐enzymatic acetyl phosphate‐dependent mechanism. To assess the functional significance of this post‐translational modification, we first grew wild‐type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA , a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label‐free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose‐regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate‐regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate‐dependent acetylation is a response to carbon flux that could regulate central metabolism. Using immunochemistry and label‐free, quantitative proteomics, we monitored protein lysine acetylation over time in response to glucose supplementation. The resultant data supports the hypothesis that carbon flux in excess of central metabolic capacity (overflow metabolism) causes acetyl phosphate‐dependent acetylation and the hypothesis that the major carbon regulator CRP regulates this process by facilitating the synthesis of acetyl phosphate.
Recent studies on CRISPR ‐based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon S ulfolobus islandicus REY 15 A encodes three interference modules, one of type IA and two of type IIIB . Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference‐mediated by one type IIIB module Cmr ‐α, and a Csx 1 protein, efficiently eliminated plasmid vectors carrying matching protospacers but lacking PAM motifs. Moreover, Cmr ‐α‐mediated interference was dependent on directional transcription of the protospacer, in contrast to the transcription‐independent activities of the type IA and type IIIA DNA interference. We infer that the interference mechanism involves transcription‐dependent DNA targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S . islandicus REY 15 A which cooperate functionally by sharing a single Cas 6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.
P>The ability of a bacterial cell to monitor and adaptively respond to its environment is crucial for survival. After one- and two-component systems, extracytoplasmic function (ECF) sigma factors - the largest group of alternative sigma factors - represent the third fundamental mechanism of bacterial signal transduction, with about six such regulators on average per bacterial genome. Together with their cognate anti-sigma factors, they represent a highly modular design that primarily facilitates transmembrane signal transduction. A comprehensive analysis of the ECF sigma factor protein family identified more than 40 distinct major groups of ECF sigma factors. The functional relevance of this classification is supported by the sequence similarity and domain architecture of cognate anti-sigma factors, genomic context conservation, and potential target promoter motifs. Moreover, this phylogenetic analysis revealed unique features indicating novel mechanisms of ECF-mediated signal transduction. This classification, together with the web tool ECFfinder and the information stored in the Microbial Signal Transduction (MiST) database, provides a comprehensive resource for the analysis of ECF sigma factor-dependent gene regulation.
All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX‐dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX . We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double‐tryptophan‐containing sigX ‐inducing peptide (XIP) pheromone. In all three groups, the XIP gene ( comS ), and sigX have identical, non‐canonical promoters consisting of 9 bp inverted repeats separated from a −10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD , suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C‐terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans , and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.
Biofilms are surface‐associated bacterial aggregates, in which bacteria are enveloped by polymeric substances known as the biofilm matrix. Bacillus subtilis biofilms display persistent resistance to liquid wetting and gas penetration, which probably explains the broad‐spectrum resistance of the bacteria in these biofilms to antimicrobial agents. In this study, BslA (formerly YuaB) was identified as a major contributor to the surface repellency of B. subtilis biofilms. Disruption of bslA resulted in the loss of surface repellency and altered the biofilm surface microstructure. BslA localized to the biofilm matrix in an exopolysaccharide‐dependent manner. Purified BslA exhibited amphiphilic properties and formed polymers in response to increases in the area of the air–water interface in vitro . Genetic and biochemical analyses showed that the self‐polymerization activity of BslA was essential for its ability to localize to the biofilm matrix. Confocal laser scanning microscopy showed that BslA formed a layer on the biofilm surface. Taken together, we propose that BslA, standing for biofilm‐surface layer protein, is responsible for the hydrophobic layer on the surface of biofilms.
P>Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5' and 3' untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.
All free‐living bacteria carry the toxin–antitoxin (TA) systems controlling cell growth and death under stress conditions. YeeU–YeeV (CbtA) is one of the Escherichia coli TA systems, and the toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. Here, we demonstrate that the antitoxin, YeeU, is a novel type of antitoxin (type IV TA system), which does not form a complex with CbtA but functions as an antagonist for CbtA toxicity. Specifically, YeeU was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro . Surprisingly, YeeU neutralized not only the toxicity of CbtA but also the toxicity caused by other inhibitors of MreB and FtsZ, such as A22, SulA and MinC, indicating that YeeU‐induced bundling of MreB and FtsZ has an intrinsic global stabilizing effect on their homeostasis. Here we propose to rename YeeU as CbeA for cytoskeleton bundling‐enhancing factor A.
The Bacillus subtilis extracytoplasmic function (ECF) σ factor σ M is inducible by, and confers resistance to, several cell envelope‐acting antibiotics. Here, we demonstrate that σ M is responsible for intrinsic β‐lactam resistance, with σ X playing a secondary role. Activation of σ M upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta‐lactam cefuroxime. However, σ M still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of σ M in β‐lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP ( yybT ) which encodes a cyclic‐di‐AMP phosphodiesterase (PDE). Intriguingly, σ M is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to β‐lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c‐di‐AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF σ factors in counteracting the deleterious effects of autolysins and reactive oxygen species in β‐lactam‐treated cells.
P>Besides industrially produced gibberellins (GAs), Fusarium fujikuroi is able to produce additional secondary metabolites such as the pigments bikaverin and neurosporaxanthin and the mycotoxins fumonisins and fusarin C. The global regulation of these biosynthetic pathways is only poorly understood. Recently, the velvet complex containing VeA and several other regulatory proteins was shown to be involved in global regulation of secondary metabolism and differentiation in Aspergillus nidulans. Here, we report on the characterization of two components of the F. fujikuroi velvet-like complex, FfVel1 and FfLae1. The gene encoding this first reported LaeA orthologue outside the class of Eurotiomycetidae is upregulated in Delta Ffvel1 microarray-studies and FfLae1 interacts with FfVel1 in the nucleus. Deletion of Ffvel1 and Fflae1 revealed for the first time that velvet can simultaneously act as positive (GAs, fumonisins and fusarin C) and negative (bikaverin) regulator of secondary metabolism, and that both components affect conidiation and virulence of F. fujikuroi. Furthermore, the velvet-like protein FfVel2 revealed similar functions regarding conidiation, secondary metabolism and virulence as FfVel1. Cross-genus complementation studies of velvet complex component mutants between Fusarium, Aspergillus and Penicillium support an ancient origin for this complex, which has undergone a divergence in specific functions mediating development and secondary metabolism.
The Bacillus subtilis extracytoplasmic function (ECF) s factor sM is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that sM is responsible for intrinsic beta-lactam resistance, with sX playing a secondary role. Activation of sM upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, sM still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of sM in beta-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, sM is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to beta-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF s factors in counteracting the deleterious effects of autolysins and reactive oxygen species in beta-lactam-treated cells.
In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base‐pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli , the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR , encoding a transcription regulator and ydaL , encoding a small MutS‐related protein. We show that McaS is a ∼ 95 nt transcript whose expression increases over growth, peaking in early‐to‐mid stationary phase, or when glucose is limiting. McaS uses three discrete single‐stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD , the transcription regulator of curli biogenesis and activates flhD , the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA , a porin required for the export of the polysaccharide poly β‐1,6‐ N ‐acetyl‐ d ‐glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.
Reactive oxygen species (ROS) are critical components of the antimicrobial repertoire of macrophages, yet the mechanisms by which ROS damage bacteria in the phagosome are unclear. The NADH‐dependent phagocytic oxidase produces superoxide, which dismutes to form H 2 O 2 . The Barras and Méresse labs use a GFP fusion to an OxyR regulated gene to show that phagocyte‐derived H 2 O 2 is gaining access to the Salmonella cytoplasm. However, they have also shown previously that Salmonella has redundant systems to detoxify this H 2 O 2 . Although Salmonella propagate in a unique vacuole, their data suggest that ROS are not diminished in this modified phagosome. These recent results are put into the context of our overall understanding of potential oxidative bacterial damage occurring in macrophages.