No abstract

Pseudomonas aeruginosa is generally described as ubiquitous in natural settings, such as soil and water. However, because anecdotal observations and published reports have questioned whether or not this description is true, we undertook a rigorous study using three methods to investigate the occurrence of P. aeruginosa: We investigated environmental samples, analyzed 16S rRNA data, and undertook a systematic review and meta‐analysis of published data. The environmental sample screening identified P. aeruginosa as significantly associated with hydrocarbon and pesticide‐contaminated environments and feces, as compared to uncontaminated environments in which its prevalence was relatively low. The 16S rRNA data analysis showed that P. aeruginosa sequences were present in all habitats but were most abundant in samples from human and animals. Similarly, the meta‐analysis revealed that samples obtained from environments with intense human contact had a higher prevalence of P. aeruginosa compared to those with less human contact. Thus, we found a clear tendency of P. aeruginosa to be present in places closely linked with human activity. Although P. aeruginosa may be ubiquitous in nature, it is usually scarce in pristine environments. Thus, we suggest that P. aeruginosa should be described as a bacterium largely found in locations associated with human activity.

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO x biosensor. The apparent halfsaturation constant [K m(app) ] value of the NH 3 oxidation of N. briensis was 3 M NH 3 for cultures grown both in continuous and batch cultures as determined by a NO x biosensor. Cells grown on the wall of the vessel had a lower K m(app) value of 1.8 M NH 3 . Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 M N h ؊1 , and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH 4 ؉ , 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammoniaoxidizing activity after addition of NH 4 ؉ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH 4 ؉ availability.Chemolithoautotrophic ammonia-oxidizing bacteria (AOB) generate their energy by oxidizing ammonia (NH 3 ) to nitrite (NO 2 Ϫ ) and fix carbon via the Calvin cycle (3, 53). The oxidation of NH 3 to NO 2 Ϫ is a two-step process, where NH 3 is first oxidized to hydroxylamine (NH 2 OH) catalyzed by ammonia monooxygenase. The NH 2 OH is further oxidized to NO 2 Ϫ catalyzed by the hydroxylamine oxidoreductase, which is the energy-generating step of the ammonia oxidation (3, 53). AOB often live in close proximity to nitrite-oxidizing bacteria and together they convert the most reduced form of nitrogen (NH 4 ϩ ) to the most oxidized (NO 3 Ϫ ) (40). In nature, AOB often face longer periods of NH 4 ϩ starvation and limitation due to low nitrogen input, low mineralization rates, or competition with other AOB (8), heterotrophic bacteria (48, 49), or plants (5,6,50). In order to respond rapidly when NH 4 ϩ becomes available, AOB must maintain their ability to oxidize NH 4 ϩ during these periods. With the exception of a few marine strains within the genus Nitrosococcus (of the ␥-subclass of the Proteobacteria), all known AOB belong to a distinct clade within the ␤-subclass of the Proteobacteria (13), which comprises 11 clusters (37). By using 16S rRNA gene and more recently amoA gene sequencing, directly from environmental samples, the distribution of the members of the different clusters of AOB has been correlated to the characteristics of the environments (29, 37). The starvation behavior of several AOB belonging to different phylogenetic groups ...

A modified protocol for simultaneous extraction of RNA and DNA, followed by real-time polymerase chain reaction quantification, was used to investigate tfdA gene expression during in situ degradation of the herbicide MCPA (4-chloro-2-methylphenoxy-acetic acid) in soil. tfdA encodes an alpha-ketoglutarate-dependent dioxygenase catalysing the first step in the degradation pathway of MCPA and 2,4-D (2,4-dichlorophenoxy-acetic acid). A linear recovery of tfdA mRNA over three orders of magnitude was shown, and the tfdA mRNA level was normalized using the tfdA mRNA/DNA ratio. The density of active cells required for tfdA mRNA detection was 10(5) cells g(-1) soil. Natural soil microcosms inoculated with Cupriavidus necator (formerly Ralstonia eutropha) AEO106 (pRO101) cells were amended with four different MCPA concentrations (2, 20, 50 and 150 mg kg(-1)). Mineralization rates were estimated by quantification of 14CO2 emission from degradation of 14C-MCPA. tfdA mRNA was detected 1 h after amendment at all four concentrations. In soils amended with 2 and 20 mg kg(-1), the mRNA/DNA ratio for tfdA demonstrated a sharp transient maximum of tfdA expression from no to full expression within 3 and 6 h respectively, followed by a decline and complete loss of expression after 19 and 43 h. A more complex pattern of tfdA expression was observed for the higher 50 and 150 mg kg(-1) amendments; this coincided with growth of C. necator AEO106 (pRO101) in the system. Repeated amendment with MCPA after 2 weeks in the 20 mg kg(-1) scenario revealed a sharp increase of tfdA mRNA, and absence of a mineralization lag phase. For all amendments, tfdA mRNA was detectable only during active mineralization, and thus revealed a direct correlation between tfdA mRNA presence and microbial degrader activity. The present study demonstrates that direct analysis of functional gene expression dynamics by quantification of mRNA can indeed be made in natural soil.