Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na ؉ /P i antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies. The Roseobacter clade is one of the most abundant groups of bacteria in oceans. The ecological success of the Roseobacter clade can be attributed to its broad metabolic capabilities (1, 2). One of the model organisms of the Roseobacter clade is Dinoroseobacter shibae. It is a mixotrophic bacterium that can utilize various organic carbon sources, including several carboxylic acids, glucose, glycerol, and succinate (1-3). Fluxome analyses showed that D. shibae lacks phosphofructokinase activity during growth on glucose and preferentially uses the Entner-Doudoroff pathway instead of glycolysis to metabolize sugar (4). Moreover, D. shibae can gain additional energy by aerobic anoxygenic photosynthesis but is unable to grow photoautotrophically. Annotation of the 4.4-Mb genome of D. shibae DFL12 T discovered genes that indicated the use of alternative electron acceptors such as nitrate and dimethyl sulfoxide in the absence of molecular oxygen (5). In agreement, anaerobic growth by denitrification was reported recently ...

Long-term catheterization inevitably leads to a catheterassociated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro-and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surfaceexposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. Molecular & Cellular Proteomics 14: 10.1074/mcp.M114.043463, 989-1008, 2015.Catheter-associated urinary tract infections (CAUTIs) 1 account for up to 40% of all nosocomial infections and are thus the most prevalent source of hospital-acquired infectious diseases (1, 2). CAUTIs are mostly "asymptomatic" and characterized by less than 10 5 colony-forming units per milliliter urine, which do not cause any signs of infection or symptoms. A symptomatic CAUTI, usually correlated to a number of colony-forming units (CFUs) exceeding the above mentioned threshold, is diagnosed when symptoms commonly associated with urinary tract infections (e.g. fever, dysuria, urgency, flank pain, or leukocytosis) occur (3). The risk that CAUTIs become symptomatic increases dramatically during catheterFrom the ‡Institute

Two strains of gram-negative bacteria isolated because of their abilities to decompose xenobiotic compounds were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence analysis, the two strains were found to belong to the genus Pseudomonas. Benzene degrading strain DSM 8628(T) was moderately related to P. flavescens NCPP 3063(T) (98.3% similarity), P. monteilii CIP 104883(T), and P. plecoglossicida FPC 951(T) (98.1%). Strain DSM 9751(T) capable to grow with cetyltrimethylammonium chloride as the sole carbon source showed the highest similarity values with P. tremae CFBP 2341(T) and P. meliae MAFF 301463(T) (98.0%), both related to Pseudomonas syringae. The fatty acid pattern of strain DSM 8628(T) was distinct from patterns of other members of the genus Pseudomonas in combining a high ratio of 3OH-C(12:1) (5.1%), a low ratio of 2OH-C(12:0) (0.2%) and a relatively low ratio of C(18:1)omega7c (23.8%). On the basis of phylogenetic analysis, physiological properties and the composition of whole cell fatty acids, two novel species, Pseudomonas benzenivorans sp. nov. with the type strain DSM 8628(T) (=CIP 109857(T)) and Pseudomonas saponiphila sp. nov. with the type strain DSM 9751(T) (=CIP 109856(T)), are proposed.

Myroides sp. A21, isolated from a urethral catheterized patient without symptoms of a urinary tract infection in Germany, proved to be extensively drug resistant. Here, we report the 4.16-Mb complete genome sequence of strain A21, carrying unusual pathogenicity islands and explaining the features of multidrug resistance.

During operation of mobile air conditioning (MAC) systems in automobiles, malodours can occur. We studied the microbial communities found on contaminated heat exchanger fins of 45 evaporators from car MAC systems which were operated in seven different regions of the world and identified corresponding volatile organic compounds. Collected biofilms were examined by scanning electron microscopy and fluorescent in situ hybridization. The detected bacteria were loosely attached to the metal surface. Further analyses of the bacteria using PCR-based single-strand conformation polymorphism and sequencing of isolated 16S rRNA gene fragments identified highly divergent microbial communities with multiple members of the Alphaproteobacteriales, Methylobacteria were the prevalent bacteria. In addition, Sphingomonadales, Burkholderiales, Bacillales, Alcanivorax spp. and Stenotrophomonas spp. were found among many others depending on the location the evaporators were operated. Interestingly, typical pathogenic bacteria related to air conditioning systems including Legionella spp. were not found. In order to determine the nature of the chemical compounds produced by the bacteria, the volatile organic compounds were examined by closed loop stripping analysis and identified by combined gas chromatography/mass spectrometry. Sulphur compounds, i.e. di-, tri- and multiple sulphides, acetylthiazole, aromatic compounds and diverse substituted pyrazines were detected. Mathematical clustering of the determined microbial community structures against their origin identified a European/American/Arabic cluster versus two mainly tropical Asian clusters. Interestingly, clustering of the determined volatiles against the origin of the corresponding MAC revealed a highly similar pattern. A close relationship of microbial community structure and resulting malodours to the climate and air quality at the location of MAC operation was concluded.