Species-specific detection of hydrocarbon-utilizing bacteria

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.     

Vitamin requirements of hydrocarbon-utilizing soil bacteria

The numbers of oil-utilizing bacteria in several samples of clean and oil-polluted soils counted on vitamin-containing media were severalfold higher than the numbers counted on vitamin-free media. Colonies that grew on a medium containing a vitamin mixture were tested for growth on the same medium lacking any vitamins. More than 90% of the total colonies failed to grow. The remaining 10% grew, yet their growth was enhanced, when vitamins were added. The predominant oil-utilizing bacteria in one of the test desert soil samples were various strains of Cellulomonas flavigena and Rhodococcus erythropolis. Minor organisms belonged to the genera Pseudomonas, Bacillus and Arthrobacter. Two vitamin-requiring biovars of C. flavigena and R. erythropolis were selected for further study. Their growth on n-octadecane and phenanthrene as sole sources of carbon and energy as well as their potential for hydrocarbon consumption were enhanced by added vitamins, e.g. folic acid, pyridoxine, vitamin B12, biotin and others. In a field experiment, it was confirmed that vitamin fertilization of an oil-polluted sand sample enhanced the biodegradation of constituent hydrocarbons of that sample.     

Peptidoglycan in obligate intracellular bacteria

Peptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.     

Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf

Green animate materials from the intertidal zone of the Arabian Gulf coast accommodated more alkaliphilic and halophilic bacteria than inanimate materials. The alkaliphilic oil-utilizing bacteria, as identified by their 16S ribonucleic acid sequences, belonged to the following genera arranged in decreasing frequences: Marinobacter, Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil-utilizing bacteria belonged to the genera: Marinobacter, Georgenia, Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. Most isolates could grow on a wide range of pure n-alkanes and aromatic compounds, as sole sources of carbon and energy. Quantitative gas liquid chromatographic analysis showed that individual isolates attenuated crude oil and representative pure hydrocarbons in culture. The optimum pH for most of the alkaliphilic genera was pH 10, and the optimum salinity for the halophiles ranged between 2.5 and 5% NaCl (w/v). It was concluded that as far as their microbial makeup is concerned, oily alkaline and saline intertidal areas of the Kuwaiti coasts have a self-cleaning potential.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

Mobile DNA in obligate intracellular bacteria

The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture.     

Metabolic interdependence of obligate intracellular bacteria and their insect hosts.

Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.     

Assimilation of chlorinated alkanes by hydrocarbon-utilizing fungi.

The fatty acid compositions of two filamentous fungi (Cunninghamella elegans and Penicillium zonatum) and a yeast (Candida lipolytica) were determined after the organisms were grown on 1-chlorohexadecane or 1-chlorooctadecane. These organisms utilized the chlorinated alkanes as sole sources of carbon and energy. Analyses of the fatty acids present after growth on the chlorinated alkanes indicated that 60 to 70% of the total fatty acids in C. elegans were chlorinated. Approximately 50% of the fatty acids in C. lipolytica were also chlorinated. P. zonatum contained 20% 1-chlorohexadecanoic acid after growth on either substrate but did not incorporate C18 chlorinated fatty acids.     

Analysis of DNA-DNA homologies in obligate methylotrophic bacteria

The genotypic affinity of 19 bacterial strains obligately dependent on methanol or methylamine as carbon and energy sources was studied by techniques of molecular DNA hybridization. The high homology level (35-88%) between motile strain Methylophilus methanolovorus V-1447D and nonmotile strain Methylobacillus sp. VSB-792 as well as other motile strains (Pseudomonas methanolica ATCC 21704, Methylomonas methanolica NRRL 5458, Pseudomonas sp. W6, strain A3) indicates that all of them belong to one genus. Rather high level of homology (62-63%) was found between Methylobacillus glycogenes ATCC 29475 and Pseudomonas insueta ATCC 21276 and strain G-10. The motile strain Methylophilus methylotrophus NCIB 10515 has a low homology (below 20%) to other of the studied obligate methylobacteria. Therefore, at least two genetically different genera of obligate methylobacteria can be distinguished, namely Methylophilus and Methylobacillus, the latter being represented by both motile and nonmotile forms.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Thermus ruber obligate thermophilic bacteria in the thermal springs of Kamchatka]

The new species of the obligate-thermophilic bacterium Thermus ruber is widely distributed in hot springs of Kamchatka with the temperature of water from 57 to 90 degrees C. The bacterium does not oxidize sulphur. It contains a red carotenoid pigment similar to neuro-sporaxanthine and retrodehydro-gamma-carotene.     

The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone