- Volume 161, Issue 1, 2015

The ECF (extracytoplasmic function) alternative sigma factor, σ22 (AlgT/U), is required for expression of the algD promoter of the operon for alginate biosynthesis in Pseudomonas aeruginosa. Alginate production promotes chronic pulmonary infections by this opportunistic pathogen in patients with cystic fibrosis and chronic obstructive pulmonary disease. σ22 is normally sequestered, but its deregulation and activation occur either by mutation in mucA (encoding an anti-sigma factor) or in response to envelope stress, such as inhibition of peptidoglycan synthesis. The σ22 stress response system includes many genes in addition to those for alginate. In the present study, we characterized an intergenic region between ORFs PA2559 and PA2560 in PAO1 for a σ22-dependent, stress-responsive transcript, described here as PA2559.1. Northern analysis and transcript end-mapping indicated the PA2559.1 transcript was ~310 nt in length. Examination of the DNA sequence upstream of +1 revealed a σ22 core promoter motif, GAATTT-N16-TCTGT, and site-directed mutagenesis confirmed this to be a σ22-dependent promoter that was highly activated during cell wall stress. PA2559.1 also contained an ORF that demonstrated increased translational activity upon cell wall stress. As determined by mutant analysis, the protein encoded by PA2559.1 was shown to play a positive role in the σ22-dependent activation of the algD promoter under stress in both sessile (i.e. biofilm) and planktonic conditions. Thus, it appeared to act as a stress response facilitator and so was named SrfA. The sequence of SrfA was found to be novel in nature and extremely well conserved only in P. aeruginosa, suggesting that it is of high evolutionary importance in this species.

In this study, the regulatory mechanism of the ula (utilization of l-ascorbic acid) operon, putatively responsible for transport and utilization of ascorbic acid in Streptococcus pneumoniae strain D39, is studied. β-Galactosidase assay data demonstrate that expression of the ula operon is increased in the presence of ascorbic acid as compared with the effects of other sugar sources including glucose. The ula operon consists of nine genes, including a transcriptional regulator UlaR, and is transcribed as a single transcriptional unit. We demonstrate the role of the transcriptional regulator UlaR as a transcriptional activator of the ula operon in the presence of ascorbic acid and show that activation of the ula operon genes by UlaR is CcpA-independent. Furthermore, we predict a 16 bp regulatory site (5′-AACAGTCCGCTGTGTA-3′) for UlaR in the promoter region of ulaA. Deletion of the half or full UlaR regulatory site in PulaA confirmed that the UlaR regulatory site present in PulaA is functional.

Marine sponges harbour abundant and diverse bacterial communities, providing an ideal environment for bacterial cell-density-dependent cell–cell signalling, termed quorum sensing. The marine sponge symbiont Ruegeria sp. KLH11 produces mainly long chain acylhomoserine lactones (AHLs) and has been developed as a quorum sensing model for roseobacterial sponge symbionts. Two pairs of luxR/I homologues were identified by genetic screening and were designated ssaRI and ssbRI (sponge-associated symbiont locus A or B, luxR/luxI homologue). In this study, we identified a third luxI-type gene, named sscI. The sscI gene does not have a cognate luxR homologue present at an adjacent locus and thus sscI is an AHL synthase solo. The sscI gene is required for production of long-chain hydroxylated AHLs, contributes to AHL pools and modestly influences flagellar motility in KLH11. A triple mutant for all luxI-type genes cannot produce AHLs, but still synthesizes para-coumaroyl-homoserine lactone.

Staphylococcus aureus is a prominent human pathogen and is known to form L-forms in vitro and in vivo during infection. However, the conditions of L-form formation are not optimal and the mechanisms of L-form formation in this organism are unknown. Here, we optimized the conditions of S. aureus unstable L-form formation, constructed a transposon mutant library and screened for mutants defective in unstable L-form formation. Our results revealed that 20 % sucrose, 3.5 % sodium chloride, 750–1000 U penicillin and 33 °C were optimal conditions for L-form formation. Stationary phase cultures of S. aureus formed L-forms better than exponential phase cultures. The S. aureus L-form colonies showed typical ‘fried-egg’ morphology and the cells had deficient cell wall, showed morphological diversity, and stained Gram-negative. The mutant library screens identified 15 mutants deficient in L-form formation and sequencing analysis identified mutations in eight genes and three intergenic regions. Real-time PCR analysis indicated that, with the exception of gntK, seven genes including glpF, glpK, NWMN_0623, NWMN_0843, NWMN_0333, NWMN_0872 and NWMN_1269 were preferentially expressed in L-forms as compared with normal cell-walled form (P<0.05). The identified genes involved in L-form growth mapped in the pathways for energy production, iron homeostasis, transporters, DNA repair, membrane biogenesis, and biosynthesis. Our findings shed new insight into the molecular basis of S. aureus unstable L-form formation and may have implications for development of novel drugs targeting S. aureus L-forms for improved treatment.

NusG, a well-conserved protein in all the three forms of life, is involved in transcription elongation and termination, as well as in the process of transcription–translation coupling. The existence of species-specific functional, as well as conformational, divergences in NusG makes it an attractive transcription factor to study, especially if it originates from a pathogen. Here, we report functional and conformational characterizations of the Mycobacterium tuberculosis (Mtb) protein Rv0639 that has been annotated as a homologue of Escherichia coli NusG. Rv0639 failed to complement the in vivo functions of E. coli NusG (Ec NusG) and did not exhibit any signature of a transcription elongation–termination factor. However, it retained the ability to bind to its cognate ribosomal protein S10 (Rv0700). Compared with Ec NusG, Rv0639 possesses unique conformational features characterized by altered secondary structures in the C-terminal domain (CTD), an unusually long and disordered linker region between the N-terminal domain (NTD) and CTD, and a folding of its NTD over its CTD. This unusual folded conformation could have imparted specialized functions to this protein, required to adapt the physiology of Mtb. We speculate that in the absence of a bona fide RfaH, a NusG paralogue that is involved in pathogenicity in E. coli, Rv0639 functions as an RfaH-like factor and is involved in pathogenicity using unidentified ops-like sequences in the Mtb genome. And hence, we reannotate Rv0639 as a paralogue of NusG, instead of a homologue.

Cyanobacteria use a sophisticated system of pigments to collect light energy across the visible spectrum for photosynthesis. The pigments are assembled in structures called phycobilisomes, composed of phycoerythrocyanin, phycocyanin and allophycocyanin, which absorb energy and transfer it to chlorophyll in photosystem II reaction centres. All of the components of this system are fluorescent, allowing sensitive measurements of energy transfer using single cell confocal fluorescence microscopy. The native pigments can be interrogated without the use of reporters. Here, we use confocal fluorescence microscopy to monitor changes in the efficiency of energy transfer as single cells age, between the time they are born at cell division until they are ready to divide again. Alteration of fluorescence was demonstrated to change with the age of the cyanobacterial cell.

Members of the SNARE protein family participate in the docking–fusion step of several intracellular vesicular transport events. Saccharomyces cerevisiae Vam7p was identified as a SNARE protein that acts in vacuolar protein transport and membrane fusion. However, in Schizosaccharomyces pombe, there have been no reports regarding the counterpart of Vam7p. Here, we found that, although the SPCC594.06c gene has low similarity to Vam7p, the product of SPCC594.06c has a PX domain and SNARE motif like Vam7p, and thus we designated the gene Sch. pombe vsl1 + (Vam7-like protein 1). The vsl1Δ cells showed no obvious defect in vacuolar protein transport. However, cells of the vsl1Δ mutant with a deletion of fsv1 +, which encodes another SNARE protein, displayed extreme defects in vacuolar protein transport and vacuolar morphology. Vsl1p was localized to the vacuolar membrane and prevacuolar compartment, and its PX domain was essential for proper localization. Expression of the fusion protein GFP-Vsl1p was able to suppress ZnCl2 sensitivity and the vacuolar protein sorting defect in the fsv1Δ cells. Moreover, GFP-Vsl1p was mislocalized in a pep12Δ mutant and in cells overexpressing fsv1 +. Importantly, overexpression of Sac. cerevisiae VAM7 could suppress the sensitivity to ZnCl2 of vsl1Δ cells and the vacuolar morphology defect of vsl1Δfsv1Δ cells in Sch. pombe. Taken together, these data suggest that Vsl1p and Fsv1p are required for vacuolar protein transport and membrane fusion, and they function cooperatively with Pep12p in the same membrane-trafficking step.

Sulfur makes up 1 % of the dry mass of bacteria, and it is an abundant element (0.1 %) on earth. Sulfur in the environment is, however, mostly in oxidized forms and inaccessible to living organisms. At present, the entire assimilation pathway of external sulfur to sulfur-containing biomolecules and its regulation in Escherichia coli remain poorly understood, except for the metabolic pathway of cysteine synthesis, the first-step metabolite of sulfur assembly. During the search for regulation targets of uncharacterized transcription factors by Genomic SELEX screening, we found that the hitherto uncharacterized YdcN regulates a set of genes involved in the utilization of sulfur, including the generation of sulfate and its reduction, the synthesis of cysteine, the synthesis of enzymes containing Fe–S as cofactors, and the modification of tRNA with use of sulfur-containing substrates. Taking these findings together, we propose renaming YdcN as SutR (regulator of sulfur utilization).

Proteins secreted by Bacillus amyloliquefaciens FZB42, a root-associated plant growth-promoting rhizobacterium, are thought to play an important role in the establishment of beneficial interactions with plants. To investigate the possible role of proteins in this process, extracellular proteome maps of B. amyloliquefaciens FZB42 during the late exponential and stationary growth phases were generated using 2D gel electrophoresis. Out of the 121 proteins identified by MALDI-TOF MS, 61 were predicted to contain secretion signals. A few of the others, bearing no signal peptide, have been described as elicitors of plant innate immunity, including flagellin proteins, cold-shock proteins and the elongation factor Tu, suggesting that B. amyloliquefaciens FZB42 protects plants against disease by eliciting innate immunity. Our reference maps were used to monitor bacterial responses to maize root exudates. Approximately 34 proteins were differentially secreted in response to root exudates during either the late exponential or stationary phase. These were mainly involved in nutrient utilization and transport. The protein with the highest fold change in the presence of maize root exudates during the late exponential growth phase was acetolactate synthase (AlsS), an enzyme involved in the synthesis of the volatile acetoin, known as an inducer of systemic resistance against plant pathogens and as a trigger of plant growth.

Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499–RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide-rich media. In order to identify functional partners to the operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared with the WT strain. This work has identified a potential interaction between RL0936 and the RL3499–RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously uncharacterized conserved hypothetical gene.

Type II toxin–antitoxin systems (TAs) are bicistronic operons ubiquitous in prokaryotic genomes, displaying multilevel association with cell physiology. Various possible functions have been assigned to TAs, ranging from beneficial for their hosts, such as a stress response, dormancy and protection against genomic parasites, to detrimental or useless functions, such as selfish alleles. As there is a link between several Escherichia coli features (e.g. virulence, lifestyle) and the phylogeny of this species, we hypothesized a similar association with TAs. Using PCR we studied the distribution of 15 chromosomal and plasmidic type II TA loci in 84 clinical E. coli isolates in relation to their main phylogenetic groups (A, B1, B2 and D). In addition, we performed in silico searching of these TA loci in 60 completely sequenced E. coli genomes deposited in GenBank. The highest number of TA loci per strain was observed in group A (mean 8.2, range 5–12) and the lowest in group B2 (mean 4.2, range 2–8). Moreover, significant differences in the prevalence of nine chromosomal TAs among E. coli phylogroups were noted. In conclusion, the presence of some chromosomal TAs in E. coli is phylogroup-related rather than a universal feature of the species. In addition, their limited collection in group B2 clearly distinguish it from the other E. coli phylogroups.

There is a growing appreciation that microbiota composition can significantly affect host health and play a role in disease onset and progression. This study assessed the impact of streptozotocin (STZ)-induced type-1-diabetes (T1D) on intestinal microbiota composition and diversity in Sprague–Dawley rats, compared with healthy controls over time. T1D was induced by injection of a single dose (60 mg STZ kg−1) of STZ, administered via the intraperitoneal cavity. Total DNA was isolated from faecal pellets at weeks 0 (pre-STZ injection), 1, 2 and 4 and from caecal content at week 5 from both healthy and T1D groups. High-throughput 16S rRNA sequencing was employed to investigate intestinal microbiota composition. The data revealed that although intestinal microbiota composition between the groups was similar at week 0, a dramatic impact of T1D development on the microbiota was apparent post-STZ injection and for up to 5 weeks. Most notably, T1D onset was associated with a shift in the Bacteroidetes : Firmicutes ratio (P<0.05), while at the genus level, increased proportions of lactic acid producing bacteria such as Lactobacillus and Bifidobacterium were associated with the later stages of T1D progression (P<0.05). Coincidently, T1D increased caecal lactate levels (P<0.05). Microbial diversity was also reduced following T1D (P<0.05). Principle co-ordinate analyses demonstrated temporal clustering in T1D and control groups with distinct separation between groups. The results provide a comprehensive account of how T1D is associated with an altered intestinal microbiota composition and reduced microbial diversity over time.

Bacteria have branched aerobic respiratory chains that terminate at different terminal oxidases. These terminal oxidases have varying properties such as their affinity for oxygen, transcriptional regulation and proton pumping ability. The focus of this study was a quinol oxidase encoded by cyoABCD. Although this oxidase (Cyo) is widespread among bacteria, not much is known about its role in the cell, particularly in bacteria that contain both cytochrome c oxidases and quinol oxidases. Using Rhizobium etli CFN42 as a model organism, a cyo mutant was analysed for its ability to grow in batch cultures at high (21 % O2) and low (1 and 0.1 % O2) ambient oxygen concentrations. In comparison with other oxidase mutants, the cyo mutant had a significantly longer lag phase under low-oxygen conditions. Using a cyo :: lacZ transcriptional fusion, it was shown that cyo expression in the wild type peaks between 1 and 2.5 % O2. In addition, it was shown with quantitative reverse transcriptase PCR that cyoB is upregulated approximately fivefold in 1 % O2 compared with fully aerobic (21 % O2) conditions. Analysis of the cyo mutant during symbiosis with Phaseolous vulgaris indicated that Cyo is utilized during early development of the symbiosis. Although it is commonly thought that Cyo is utilized only at higher oxygen concentrations, the results from this study indicate that Cyo is important for adaptation to and sustained growth under low oxygen.

Investigation of a series of nutrient-supplemented thixotropic gels at successive dilutions that impede the trajectories of a highly vigorous motile flagellated protist, Spironucleus vortens, provides insights into both its swimming characteristics and a means for its immobilization. The progress of movement of this organism through the solidified growth medium was monitored by the in situ reductive production of a formazan chromophore from a dissolved tetrazolium salt. The physical properties of the gels were measured using an Anton Paar rheometer. The test parameters and measurements included: angular frequency, complex viscosity, complex shear modulus, shear rate and rotational recovery. These rheological characteristics affected the forward velocity of the organism through the gels, during and after multiple resetting, information potentially useful for determination of the dynamic characteristics of flagellar movement and propulsion rates of the organism. Application to separation of single cells, individuals of distinct sizes or the differing species from mixed cultures of motile and non-motile organisms or less actively swimming species was evident. These applications can be used when isolating the parasite from the intestinal contents of its host or from faecal pellets.