- Volume 162, Issue 7, 2016

Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa.

Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased.

Franconibacter (Enterobacter, Cronobacter) pulveris bacteria share several typical characteristics with, and hence pose a challenge for the detection of, Cronobacter sakazakii, an emerging opportunistic pathogen, which can cause severe infections in neonates. A structurally variable O-specific polysaccharide (OPS) called O antigen provides the major basis for the typing of Gram-negative bacteria. We investigated the structure and genetics of the O antigen of F. pulveris G3872 (designated O1). An OPS was isolated by mild alkaline degradation of the LPS, whereas the same polysaccharide and its oligosaccharide fragments were obtained by mild acid degradation. Studies by sugar analysis and NMR spectroscopy showed that the OPS contained d-ribose, l-rhamnose (l-Rha) and a rarely occurring monosaccharide 4-deoxy-d-arabino-hexose, and the OPS structure was established. The O-antigen gene cluster of F. pulveris G3872 between JUMPStart and gnd genes includes putative genes for glycosyltransferases, ATP-binding cassette (ABC)-transporter genes wzm and wzt, and genes for the synthesis of l-Rha, but no genes for the synthesis of 4-deoxy-d-arabino-hexose. A mutation test with the wzm gene confirmed that the OPS is synthesized and exported by the ABC-transporter-dependent pathway. A trifunctional transferase was suggested to catalyse formation of two glycosidic linkages and add a methyl group to the non-reducing end of the OPS to terminate the chain elongation. A carbohydrate-binding module that presumably recognizes the terminal methyl-modified monosaccharide was found at the C-terminus of Wzt. Primers specific for F. pulveris G3872 were designed based on the wzm gene, which has potential to be used for identification and detection of the O1 serogroup.

Partition systems contribute to stable plasmid inheritance in bacteria through the active separation of DNA molecules to daughter cells, and the centromeric sequence located either upstream or downstream of canonical partition operons plays an important role in this process. A specific DNA-binding protein binds to this sequence and interacts with the motor NTPase protein to form a nucleoprotein complex. The inc18-family plasmid pSM19035 is partitioned by products of δ and ω genes, with δ encoding a Walker-type ATPase and ω encoding a DNA-binding protein. As the two genes are transcribed separately, this system differs from others in its organization; nonetheless, expression of these genes is regulated by Omega, which also regulates the copy number of the plasmid (by controlling copS gene expression). Protein Omega specifically recognizes WATCACW heptad repeats. In this study, we constructed a synthetic δω operon to enable an analysis of the centromeric functions of Omega-binding sites Pδ , Pω and PcopS , discrete from their promoter functions. Our results show that these three regions do not support plasmid stabilization equally. We demonstrate that the Pω site alone can simultaneously drive the expression of partition genes from the synthetic δω operon and act as a unique centromeric sequence to promote the most efficient plasmid partitioning. Moreover, Pω can support the centromeric function in concert with the synthetic δω operon expressed from a heterologous promoter demonstrating that Pω is the main centromeric sequence of the δ–ω partition system. Additionally, the RNA polymerase-recognized sequence in Pω is essential for its centromeric function.

A total of 218 actinobacteria strains were isolated from wild perennial liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza. inflate BAT. Based on morphological characteristics, 45 and 32 strains from G. inflate and G. glabra, respectively, were selected for further analyses. According to 16S rRNA sequence analysis, most of the strains belonged to genus Streptomyces and a few strains represented the rare actinobacteria Micromonospora, Rhodococcus and Tsukamurella. A total of 39 strains from G. inflate and 27 strains from G. glabra showed antimicrobial activity against at least one indicator organism. The range of the antimicrobial activity of the strains isolated from G. glabra and G. inflate was similar. A total of 34 strains from G. inflate and 29 strains from G. glabra carried at least one of the genes encoding polyketide synthases, non-ribosomal peptide synthetase and FADH2-dependent halogenase. In the type II polyketide synthase KSα gene phylogenetic analysis, the strains were divided into two major clades: one included known spore pigment production-linked KSα sequences and other sequences were linked to the production of different types of aromatic polyketide antibiotics. Based on the antimicrobial range, the isolates that carried different KSα types were not separated from each other or from the isolates that did not carry KSα. The incongruent phylogenies of 16S rRNA and KSα genes indicated that the KSα genes were possibly horizontally transferred. In all, the liquorice plants were a rich source of biocontrol agents that may produce novel bioactive compounds.

Pressmud is a substrate derived from sugarcane juice filtrate, and around 26–40  kg of this residue are produced per ton of sugarcane. It is mainly used as fertilizer in crops, and its application in the field is often made without any prior treatment, but, in this research, it was studied for the risk this practice poses for human health. This research was stimulated by previous results indicating the presence of opportunistic pathogens in residues used in various composting systems and the extensive use of fresh pressmud in agriculture. Here, It was assessed the fungal diversity present in both fresh and composting pressmud using 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their enzymatic repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). A wide range of opportunistic pathogens was found among the most abundant taxa in the fresh pressmud, such as Lomentospora prolificans (43.13 %), Trichosporon sp. (10.07 %), Candida tropicalis (7.91 %), and Hormographiella aspergillata (8.19 %). This indicates that fresh pressmud might be a putative source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without any treatment. With regard to the heat-tolerant fungi found in this substrate, all the 110 isolates screened were able to produce at least one of the tested enzymes. The pressmud composting process not only effectively reduces the load of pathogenic fungi, but also creates an interesting environment for fungi able to produce thermostable hydrolytic and oxidative enzymes with biotechnological applications.

Gene organization and control are described by models conceived in the 1960s. These models explain basic gene regulatory mechanisms and underpin current genome annotation. However, such models struggle to explain recent genome-scale observations. For example, accounts of RNA synthesis initiating within genes, widespread antisense transcription and non-canonical DNA binding by gene regulatory proteins are difficult to reconcile with traditional thinking. As a result, unexpected observations have often been dismissed and downstream consequences ignored. In this paper I will argue that, to fully understand the biology of bacterial chromosomes, we must embrace their hidden layers of complexity.

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.

Lactobacilli are the dominant bacteria of the vaginal tract of healthy women, and imbalance of the local microbiota can predispose women to acquire infections, such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). Although antimicrobial therapy is generally effective, there is still a high incidence of recurrence and increase of microbial resistance due to the repetitive use of antimicrobials. Thus, it has been suggested that administration of probiotics incorporating selected Lactobacillus strains may be an effective strategy for preventing vaginal infections. Accordingly, the in vitro probiotic potential of 23 lactobacilli isolated from the vaginal ecosystem of healthy women from Cuba was evaluated for use in BV and VVC treatments. Eight strains were selected based on their antagonist potential against Gardnerella vaginalis, Candida albicansor both. In vitro assays revealed that all these strains reduced the pathogen counts in co-incubation, showed excellent adhesive properties (biofilm formation and auto-aggregation), were able to co-aggregate with G. vaginalis and C. albicans, yielded high amounts of hydrogen peroxide and lactic acid and demonstrated high adhesion rates to epithelial HeLa cells. Interference tests within HeLa cells showed that all strains were able to reduce the adherence of pathogens by exclusion or displacement. Lactobacilli were able to inhibit HeLa cell apoptosis caused by pathogens when the cells were incubated with the probiotics prior to challenge. These results suggest that these strains have a promising probiotic potential and can be used for prevention or treatment of BV and VVC.

Two homologous proteins, UxuR and ExuR, were previously predicted to repress synthesis of enzymes required for hexuronic acid metabolism, but little is known about the relative roles of these proteins in gene regulation. We confirmed the previous report that UxuR is essential for rapid growth with d-glucuronate as the primary source of carbon and energy. In contrast, an exuR mutant grew more rapidly on d-glucuronate than the parent. Transcription of exuR is initiated at a σ70–dependent promoter predicted in silico. Purified ExuR bound to the exuR regulatory region in the presence, but not in the absence, of d-glucuronate. Apparently weaker UxuR binding in the presence of glucuronate was also detected, and its addition decreased ExuR binding by forming ExuR–UxuR heterodimers. Glucuronate induced exuR transcription in the parental strain, but not in the exuR mutant. No evidence was obtained for cAMP-dependent regulation of exuR by the catabolite repressor protein (CRP). A previous study reported that the divergent yjjM and yjjN genes, essential for l-galactonate metabolism, are repressed by UxuR. We showed that ExuR binds to the yjjM-yjjN regulatory region, and that the binding is also glucuronate-dependent. As for the exuR promoter, UxuR appeared to decrease ExuR binding. ExuR is required for glucuronate induction of yjjM and yjjN, and CRP is required for their transcription. The combined data established that UxuR and ExuR fulfil contrasting roles in regulating hexuronic acid metabolism and indicate that ExuR can function as a transcription activator, possibly by inactivating the repressor function of UxuR by heterodimer formation.

Utilization of hydroxy-l-proline (l-Hyp) in Pseudomonas aeruginosa requires conversion of l-Hyp to d-Hyp followed by the d-Hyp dehydrogenase pathway; however, the molecular mechanism in control of l-Hyp catabolism and transport was not clear. DNA microarray analysis revealed twelve genes in two adjacent loci that were induced by exogenous l-Hyp and d-Hyp. The first locus includes lhpABFE encoding a Hyp epimerase (LhpA) and d-Hyp dehydrogenase (LhpBEF), while the second locus codes for a putative ABC transporter (LhpPMNO), a protein of unknown function (LhpH), Hyp/Pro racemase (LhpK) and two enzymes in l-Hyp catabolism (LhpC and LhpG). Proximal to these two loci, lhpR encodes a transcriptional regulator of the AraC family. The importance of these genes on l-Hyp catabolism was supported by growth phenotype analysis on knockout mutants. Induction of the lhpA and lhpP promoters by exogenous l-Hyp and d-Hyp was demonstrated by the measurement of β-galactosidase activities from promoter-lacZ fusions in PAO1, and no induction could be detected in the ΔlhpR mutant. Induction of the lhpA promoter by d-Hyp was completely abolished in the lhpA lhpK double mutant devoid of two epimerases, while the induction effect of l-Hyp remained unchanged. The purified His-tagged LhpR binds specifically to the lhp promoter regions, and formation of nucleoprotein complexes is affected by the presence of l-Hyp but not d-Hyp. Putative LhpR binding sites were deduced from serial deletions and comparative genomic sequence analysis. In summary, expression of lhp genes for Hyp catabolism and uptake requires the transcriptional activator LhpR and l-Hyp as the signalling compound.

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.