Developmental transcriptome of resting cell formation in Mycobacterium smegmatis.

BACKGROUND: Mycobacteria, along with exospore forming Streptomyces, belong to the phylum actinobacteria. Mycobacteria are generally believed to be non-differentiating. Recently however, we showed that the mycobacterial model organism M. smegmatis is capable of forming different types of morphologically distinct resting cells. When subjected to starvation conditions, cells of M. smegmatis exit from the canonical cell division cycle, segregate and compact their chromosomes, and become septated and multi-nucleoided. Under zero nutrient conditions the differentiation process terminates at this stage with the formation of Large Resting Cells (LARCs). In the presence of traces of carbon sources this multi-nucleoided cell stage completes cell division and separates into Small Resting Cells (SMRCs). Here, we carried out RNA-seq profiling of SMRC and LARC development to characterize the transcriptional program underlying these starvation-induced differentiation processes. RESULTS: Changes among the top modulated genes demonstrated that SMRCs and LARCs undergo similar transcriptional changes. The formation of multi-nucleoided cells (i.e. LARCs and the LARC-like intermediates observed during SMRC formation) was accompanied by upregulation of septum formation functions FtsZ, FtsW, and PbpB, as well as the DNA translocase FtsK. The observed compaction of chromosomes was accompanied by an increase of the transcript level of the DNA binding protein Hlp, an orthologue of the Streptomyces spore-specific chromosome condensation protein HupS. Both SMRC and LARC development were accompanied by similar temporal expression patterns of candidate regulators, including the transcription factors WhiB2, WhiB3, and WhiB4, which are orthologues of the Streptomyces sporulation regulators WhiB, WhiD and WblA, respectively. CONCLUSIONS: Transcriptional analyses of the development of mycobacterial resting cell types suggest that these bacteria harbor a novel differentiation program and identify a series of potential regulators. This provides the basis for the genetic dissection of this actinobacterial differentiation process.

Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.

Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp.

Pectobacterium atrosepticum (Pca) is a Gram-negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3-oxo-hexanoyl-l-homoserine lactone (OHHL), and (ii) the intracellular 'alarmone', (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence-associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.

Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity.

Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels.

Genomic variation among contemporary Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis patients.

The airways of individuals with cystic fibrosis (CF) often become chronically infected with unique strains of the opportunistic pathogen Pseudomonas aeruginosa. Several lines of evidence suggest that the infecting P. aeruginosa lineage diversifies in the CF lung niche, yet so far this contemporary diversity has not been investigated at a genomic level. In this work, we sequenced the genomes of pairs of randomly selected contemporary isolates sampled from the expectorated sputum of three chronically infected adult CF patients. Each patient was infected by a distinct strain of P. aeruginosa. Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified in the DNA common to the paired isolates from different patients. The paired isolates from one patient differed due to just 1 SNP and 8 indels. The paired isolates from a second patient differed due to 54 SNPs and 38 indels. The pair of isolates from the third patient both contained a mutS mutation, which conferred a hypermutator phenotype; these isolates cumulatively differed due to 344 SNPs and 93 indels. In two of the pairs of isolates, a different accessory genome composition, specifically integrated prophage, was identified in one but not the other isolate of each pair. We conclude that contemporary isolates from a single sputum sample can differ at the SNP, indel, and accessory genome levels and that the cross-sectional genomic variation among coeval pairs of P. aeruginosa CF isolates can be comparable to the variation previously reported to differentiate between paired longitudinally sampled isolates.

EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42.

Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 (Shigella), SifA, SifB (Salmonella) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism.