SciCombinator

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Journal: Environmental microbiology

30

We studied the vanA-carrying vancomycin-resistant enterococci (VRE) isolated from American crows in the United States during the winter 2011/2012. Faecal samples from crows were cultured selectively for VRE and characterized. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to examine epidemiological relationships of vanA-containing VRE. Isolates were tested in vitro for their ability to horizontally transfer the vancomycin resistance trait. VRE with the vanA gene were found in 15 (2.5%) of 590 crows samples, from which we obtained 22 different isolates. Enterococcal species were Enterococcus faecium (14) and E. faecalis (8). One, two and 19 isolates originated from Kansas, New York State and Massachusetts, respectively. Based on MLST analysis, E. faecium isolates were grouped as ST18 (6 isolates), ST555 (2), and novel types ST749 (1), ST750 (3), ST751 (1), ST752 (1). Enterococcus faecalis isolates belonged to ST6 (1), ST16 (3) and ST179 (4). All isolates were able to transfer the vancomycin resistance trait via filter mating with very high transfer range. Clinically important enterococci with the vanA gene occur in faeces of wild American crows throughout the United States. These migrating birds may contribute to the dissemination of VRE in environment over large distances. [Correction added after first online publication on 06 August 2013: The number of E. faecium ST752 isolate is now amended to ‘1’, consistent with that shown in the ‘Results’ section and Figure 2.].

Concepts: United States, Antibiotic resistance, Linezolid, Vancomycin, Enterococcus, Vancomycin-resistant enterococcus, Enterococcus faecium, Enterococcus faecalis

29

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected ‘Candidatus Piscichlamydia salmonis’ and ‘Candidatus Clavochlamydia salmonicola’ infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Concepts: Causality, Fish, Salmon, Salmonidae, Salmo, Trout, Brown trout, Brook trout

28

The catalase family of Beauveria bassiana (fungal entomopathogen) consists of catA (spore-specific), catB (secreted), catP (peroxisomal), catC (cytoplasmic) and catD (secreted peroxidase/catalase), which were distinguished in phylogeny and structure and functionally characterized by constructing single-gene disrupted and rescued mutants for enzymatic and multi-phenotypic analyses. Total catalase activity decreased 89% and 56% in ΔcatB and ΔcatP, corresponding to the losses of upper and lower active bands gel-profiled for all catalases respectively, but only 9-12% in other knockout mutants. Compared with wild type and complement mutants sharing similar enzymatic and phenotypic parameters, all knockout mutants showed significant (9-56%) decreases in the antioxidant capability of their conidia (active ingredients of mycoinsecticides), followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects included mainly the losses of 40% thermotolerance (45°C) in ΔcatA, 46-48% UV-B resistance in ΔcatA and ΔcatD, and 33-47% virulence to Spodoptera litura larvae in ΔcatA, ΔcatP and ΔcatD respectively. Moreover, the drastic transcript upregulation of some other catalase genes observed in the normal culture of each knockout mutant revealed functionally complimentary effects among some of the catalase genes, particularly between catB and catC whose knockout mutants displayed little or minor phenotypic changes. However, the five catalase genes functioned redundantly in mediating the fungal tolerance to either hyperosmotic or fungicidal stress. The differentiated roles of five catalases in regulating the B. bassiana virulence and tolerances to oxidative stress, high temperature and UV-B irradiation provide new insights into fungal adaptation to stressful environment and host invasion.

Concepts: Gene, Cell, Bacteria, Enzyme, Fungus, Mutant, Catalase, Beauveria bassiana

28

Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an ‘orphan’ homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic ‘fitness island’ or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a genomic island or production of extracellular filaments.

Concepts: DNA, Gene, Gene expression, Bacteria, Evolution, Cell membrane, Legionella, Legionella pneumophila

28

Pseudomonas putida KT2440 has evolved a tightly regulated system for metabolizing glycerol implying a prolonged growth lag-phase. We have learnt that this fact can be avoided by the addition of small amounts of some growth precursors. The addition of 1 mM octanoic acid as co-feeder completely eliminated the lag-phase, resulting in an improvement, in terms of invested time, of both growth and polyhydroxyalkanoates (PHA) accumulation. To investigate this phenomenon, we have followed co-metabolic approaches combined with mutations of the specific and global regulatory networks that connect glycerol catabolism and PHA synthesis. By using mutant strains in metabolic genes from the PHA and tricarboxylic acid (TCA) cycles, we have demonstrated that the co-feeding effect is independent of PHA accumulation, but driven on active glyoxylate shunt and Entner-Doudoroff (ED) routes. These findings suggested that the effect of octanoate on glycerol metabolism could rely, either on a global activation of the cell energy state, or on the generation of specific metabolites or cofactors needed for the activation of glycerol metabolism. Our results addressed GlpR as the key factor controlling the efficient utilization of glycerol as growth precursor in P. putida KT2440. Accordingly, a glpR knockout mutant of P. putida KT2440 showed an elimination of the lag-phase when cultured on glycerol in the absence of co-feeder. Besides, the production of PHA in this strain was increased near twofold, resulting in a higher final yield in terms of PHA accumulation. The repressor activity of the GlpR protein over the glp genes in the absence of glycerol was finally demonstrated by qRT-PCR. This work contributed to unravel the physiological causes of the long lag-phase produced by glycerol in the model strain P. putida KT2440 that hinders its use as carbon source in biotechnological applications for generating valuable products.

Concepts: Protein, Bacteria, Evolution, Amino acid, Metabolism, Pseudomonas, Pseudomonas putida, Polyhydroxyalkanoates

28

High-throughput identification of proteins with the latest generation of hybrid high-resolution mass spectrometers is opening new perspectives in microbiology. I present, here, an overview of tandem mass spectrometry technology and bioinformatics for shotgun proteomics that make 2D-PAGE approaches obsolete. Non-labelling quantitative approaches have become more popular than labelling techniques on most proteomic platforms because they are easier to carry out while their quantitative outcome is rather robust. Parameters for recording mass spectrometry data, however, need to be chosen carefully and statistics to assess the confidence of the results should not be neglected. Interestingly, next-generation sequencing methodologies make any microbial model quickly amenable to proteomics, leading to the documentation of a wide range of organisms from diverse environments. Some recent discoveries made using microbial proteomics have challenged some biological dogma, such as: (i) initiation of the translation does not occur predominantly from ATG codons in some microorganisms, (ii) non-canonical initiation codons are used to regulate the production of specific but important proteins and (iii) a gene may code for multiple polypeptide species, heterogeneous in terms of sequences. Microbial diversity and microbial physiology can now be revisited by means of exhaustive comparative proteomic surveys where thousands of proteins are detected and quantified. Proteogenomics, consisting of better annotating of genomes with the help of proteomic evidence, is paving the way for integrated multi-omic approaches in microbiology. Finally, meta-proteomic tools and approaches are emerging for tackling the high complexity of the microbial world as a whole, opening new perspectives for assessing how microbial communities function.

Concepts: Protein, Gene, Bioinformatics, Mass spectrometry, Organism, Tandem mass spectrometry, Top-down proteomics, Shotgun proteomics

28

The Crc protein of Pseudomonas inhibits the expression of genes involved in the transport and assimilation of a number of non-preferred carbon sources when preferred substrates are available, thus coordinating carbon metabolism. Crc acts by binding to target mRNAs, inhibiting their translation. In Pseudomonas putida, the amount of free Crc available is controlled by two sRNAs, CrcY and CrcZ, which bind to and sequester Crc. The levels of these sRNAs vary according to metabolic conditions. Pseudomonas putida grows optimally at 30°C, but can also thrive at 10°C. The present work shows that when cells grow exponentially at 10°C, the repressive effect of Crc on many genes is significantly reduced compared with that seen at 30°C. Total Crc levels were similar at both temperatures, but those of CrcZ and CrcY were significantly higher at 10°C. Therefore, Crc-mediated repression may, at least in part, be reduced at 10°C because the fraction of Crc protein sequestered by CrcZ and CrcY is larger, reducing the amount of free Crc available to bind its targets. This may help P. putida to face cold stress. The results reported might help understanding the behaviour of this bacterium in bioremediation or rhizoremediation strategies at low temperatures.

Concepts: DNA, Gene, Gene expression, Bacteria, Metabolism, Enzyme, Organism, Pseudomonas putida

28

The general stress response and the decision-making processes of sporulation initiation are interconnected pathways in the regulatory network of Bacillus subtilis. In a previous study we provided evidence for a mechanism capable of impairing sporulation by σ(B) -dependent induction of spo0E, encoding a phosphatase specifically inactivating the sporulation master regulator Spo0A∼P. Here we show that the σ(B) promoter (Pσ(B) ) of spo0E is responsive to sub-inhibitory levels of ethanol stress, producing a σ(B) -dependent sporulation deficient phenotype. In addition to positive regulation by σ(B) , we identified Rok, the repressor of comK, to be a direct repressor of spo0E expression from Pσ(B) . This constellation provides the possibility to integrate signals negatively acting on sporulation initiation through the σ(B) branch as well as a positive feedback loop acting on Pσ(B) by Rok that is most likely a direct consequence of Spo0A∼P activity. Thus, the molecular mechanism described here offers the opportunity for cross-talk between the general stress response and sporulation initiation in the adaptational gene expression network of B. subtilis.

Concepts: DNA, Gene, Gene expression, Transcription, Bacillus, Feedback, Bacillus subtilis, Endospore

28

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 μm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757(T) and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754(T) and Hfx. volcanii DSM 3757(T) , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754(T) showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

Concepts: Archaea, Bacteria, Microbiology, Microscope, Biofilm, Microscopy, Pilus, Halobacteriaceae

25

Glass sponge (Hexactinellida, Porifera) is a special lineage due to its unique tissue organization and skeleton material. Structure and physiology of glass sponge have been extensively studied. However, our knowledge of the glass sponge-associated microbial community and the interaction with the host is rather limited. Here, we performed genomic studies on the microbial community in the glass sponge Lophophysema eversa in seamount. The microbial community was dominated by an ammonia-oxidizing archaeum (AOA), a nitrite-oxidizing bacterium (NOB) and a sulfur-oxidizing bacterium (SOB), all of which were autotrophs. Genomic analysis on the AOA, NOB and SOB in the sponge revealed specific functional features of sponge-associated microorganisms in comparison to the closely related free-living relatives, including chemotaxis, phage defense, vitamin biosynthesis and nutrient uptake among others, which are related to ecological functions. The three autotrophs play essential roles in the cycles of carbon ©, nitrogen (N) and sulfur (S) in the microenvironment inside the sponge body, and they are considered to play symbiotic roles in the host as scavengers of toxic ammonia, nitrite and sulfide. Our study extends knowledge regarding the metabolism and evolution of chemolithotrophs inside the invertebrate body.

Concepts: Photosynthesis, Archaea, Bacteria, Evolution, Amino acid, Metabolism, Microbiology, Sponge