- Volume 161, Issue 10, 2015

Essential gene studies often reveal novel essential functions for genes with dispensable homologues in other species. This is the case with the widespread family of electron transfer flavoproteins (ETFs), which are required for the metabolism of specific substrates or for symbiotic nitrogen fixation in some bacteria. Despite these non-essential functions high-throughput screens have identified ETFs as putatively essential in several species. In this study, we constructed a conditional expression mutant of one of the ETFs in Burkholderia cenocepacia, and demonstrated that its expression is essential for growth on both complex media and a variety of single-carbon sources. We further demonstrated that the two subunits EtfA and EtfB interact with each other, and that cells depleted of ETF are non-viable and lack redox potential. These cells also transition from the short rods characteristic of Burkholderia cenocepacia to small spheres independently of MreB. The putative membrane partner ETF dehydrogenase also induced the same rod-to-sphere change. We propose that the ETF of Burkholderia cenocepacia is a novel antibacterial target.

Ixotrophy is a process that enables certain microbes to prey on other cells. The ability of cells to aggregate or adhere is thought to be a significant initial step in ixotrophy. The gliding, multicellular filamentous bacterium Aureispira sp. CCB-QB1 belongs to the family Saprospiraceae and preys on bacteria such as Vibrio sp. in seawater. Adhesion and cell aggregation were coincident with preying and were hypothesized to play an important role in the ixotrophy in this bacterium. To test this hypothesis, experiments to elucidate the mechanisms of aggregation or adhesion in this bacterium were performed. The ability of Aureispira QB1 to adhere and aggregate to prey bacterium, Vibrio sp., required divalent cations, especially calcium ions. In the presence of calcium, Aureispira QB1 cells captured 99 % of Vibrio sp. cells after 60 min of incubation. Toluidine blue O, which binds acidic polysaccharides, bound to Aureispira QB1 and inhibited adhesion of Aureispira QB1. These results suggest that acidic polysaccharides are needed for aggregation or adhesion of Aureispira and that calcium ions play a significant role in these phenomena.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Probiotics are bacteria used in the food industry due to their potential health benefits. In this study, the plasma membrane of the probiotic Lactobacillus acidophilus La-5 was investigated using state-of-the-art high-resolution shotgun lipidomics. Comparisons of the lipidome of the plasma membrane were done after altering the fatty acid composition by supplementing L. acidophilus La-5 with saturated, mono-, di- and tri-unsaturated fatty acids during fermentation. The plasma membrane with the highest degree of saturation resulted in a lipid composition with the highest proportion of cardiolipin (CL) and lowest proportion of monolysocardiolipin (MLCL). No significant changes were found for other lipid classes. The bacteria grown with di- and tri-unsaturated fatty acids were expected to have more unsaturated plasma membranes than bacteria grown with mono-unsaturated fatty acids. This was also the case for MLCL, but the numbers of double bonds for CL were quite similar for these three samples. The results indicate that L. acidophilus La-5 possesses a molecular mechanism for remodelling and optimizing the fatty acid composition of CL and MLCL species and the molar ratio of CL and MLCL. This study contributes new knowledge on the previously uninvestigated lipidome of L. acidophilus La-5.

Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to β-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on β-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to β-lactam-mediated inhibition of cell division, rapidly appeared upon β-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of β-lactam treatment. Interestingly, the effect of sugar on β-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in β-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.

Fructophily has been described in yeasts as the ability to utilize fructose preferentially when fructose and glucose are available in the environment. In Zygosaccharomyces bailii and Zygosaccharomyces rouxii, fructophilic behaviour has been associated with the presence of a particular type of high-capacity and low-affinity fructose transporters designated Ffz. In this study, a PCR screening was performed in several yeasts using degenerate primers suitable to detect FFZ-like genes. In parallel, fructophilic character was evaluated in the same strains by comparing the relative consumption rate of fructose and glucose. For all the strains in which FFZ-like genes were detected, fructophilic behaviour was observed (25 strains). Results show that FFZ genes are ubiquitous in the Zygosaccharomyces and Starmerella clades. Strains of Lachancea fermentati, Torulaspora microellipsoides and Zygotorulaspora florentina were not fructophilic and did not harbour FFZ genes. It is of note that these new species were recently removed by taxonomists from the Zygosaccharomyces clade, supporting the view that the presence of FFZ-like genes is a main characteristic of Zygosaccharomyces. Among the strains tested, only Hanseniaspora guilliermondii NCYC2380 was an exception, having a preference for fructose in medium with high sugar concentrations, despite no FFZ-like genes being detected in the screening. Furthermore, this study supports the previous idea of the emergence of a new family of hexose transporters (Ffz facilitators) distinct from the Sugar Porter family.

The NsrR protein of Escherichia coli is a transcriptional repressor that contains an [Fe–S] cluster that is the binding site for nitric oxide (NO). Reaction of NsrR with NO leads to de-repression of its target genes, which include those encoding an NO scavenging flavohaemoglobin and the RIC (repair of iron centres) protein involved in the repair of NO-damaged [Fe–S] clusters. The nsrR gene is promoter proximal in a transcription unit with rnr, encoding the cold shock-inducible RNase R. Here, we show that nsrR is expressed from a strong promoter, but that its translation is extremely inefficient, leading to a low cellular NsrR concentration. Conversion of the nsrR start codon from the wild-type GUG to AUG increased the efficiency of translation (which, nevertheless, remained extremely low) and had measurable effects on the expression of some NsrR-regulated genes. We conclude that NsrR abundance in the cell is such that promoters with low-affinity NsrR binding sites may partially escape NsrR-mediated repression. Expression profiling confirmed that genes regulated by NsrR (whether directly or indirectly) tend to express lower mRNA levels when the nsrR start codon is AUG than when it is GUG. Transcriptomics data implicated the pyruvate oxidase gene poxB as a novel NsrR target, which we confirmed and showed to be due to read-through transcription from the upstream hcp-hcr genes. We also present evidence to suggest that NsrR is a regulator of the sufABCDSE genes, which encode the components of an [Fe–S] cluster biogenesis and repair system.

Various phenotypes ranging from biofilm formation to pigment production have been shown to be regulated by quorum sensing (QS) in many bacteria. However, studies of the regulation of pigments produced by marine bacteria in saline conditions and of biofilm-associated phenotypes are scarcer. This study focuses on the demonstration of the existence of a QS communication system involving N-acylhomoserine lactones (AHLs) in the Mediterranean Sea strain Pseudoalteromonas ulvae TC14. We have investigated whether TC14 produces the violacein pigment, and whether intrinsic or exogenous AHLs could influence its production and modulate biofilm-associated phenotypes. Here, we demonstrate that the purple pigment produced by TC14 is violacein. The study shows that in planktonic conditions, TC14 produces more pigment in the medium in which it grows less. Using different approaches, the results also show that TC14 does not produce intrinsic AHLs in our conditions. When exogenous AHLs are added in planktonic conditions, the production of violacein is upregulated by C6-, C12-, 3-oxo-C8 and 3-oxo-C12-HSLs (homoserine lactones), and downregulated by 3-oxo-C6-HSL. In sessile conditions, 3-oxo-C8-HSL upregulates the production of violacein. The study of the biofilm-associated phenotypes shows that oxo-derived-HSLs decrease adhesion, swimming and biofilm formation. While 3-oxo-C8 and 3-oxo-C12-HSLs decrease both swimming and adhesion, 3-oxo-C6-HSLs decrease not only violacein production in planktonic conditions but also swimming, adhesion and more subtly biofilm formation. Therefore, TC14 may possess a functional LuxR-type QS receptor capable of sensing extrinsic AHLs, which controls violacein production, motility, adhesion and biofilm formation.