Pedro F. B. Brandao

Physiology, biochemistry and taxonomy of deep-sea nitrile metabolising Rhodococcus strains

A collection of nitrile-hydrolysing rhodococci was isolated from sediments sampled from a range of deep coastal, and abyssal and hadal trench sites in the NW Pacific Ocean, as part of our programme on the diversity of marine actinomycetes. Nitrile-hydrolysing strains were obtained by batch enrichments on nitrile substrates with or without dispersion and differential centrifugation pre-treatment of sediments, and were recovered from all of the depths sampled (approximately 1100–6500 m). Two isolates obtained from the Ryukyu (5425 m) and Japan (6475 m) Trenches, and identified as strains of Rhodococcus erythropolis,were chosen for detailed study. Both of the deep-sea isolates grew at in situ temperature (4°C), salinities (0–4% NaCl) and pressures (40–60 MPa), results that suggest, but do not prove, that they may be indigenous marine bacteria. However, the absence of culturable Thermoactinomycespoints to little or no run off of terrestrial microbiota into these particular trench sediments. Nitrile-hydrolysis by these rhodococci was catalysed by a nitrile hydratase–amidase system. The hydratase accommodated aliphatic, aromatic and dinitrile substrates, and enabled growth to occur on a much wider range of nitriles than the only other reported marine nitrile-hydrolysing R. erythropolis which was isolated from coastal sediments. Also unlike the latter strain, the nitrile hydratases of the deep-sea rhodococci were constitutive. The possession of novel growth and enzyme activities on nitriles by these deep-sea R. erythropolisstrains recommends their further development as industrial biocatalysts.

Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats

ABSTRACT A molecular screening approach was developed in order to amplify the genomic region that codes for the α- and β-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066T, which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.

Dereplication for biotechnology screening: PyMS analysis and PCR–RFLP–SSCP (PRS) profiling of 16S rRNA genes of marine and terrestrial actinomycetes

Abstract. The search for exploitable biology is a major task for biotechnology-based industries. In this context, discrimination between previously tested or recovered micro-organisms (dereplication) is imperative, in order to reduce screening costs by sorting large collections of isolates, which are then subjected to further detailed evaluation. Pyrolysis mass spectrometry (PyMS) is a whole-cell fingerprinting technique that enables the rapid and reproducible sorting of micro-organisms, uses small samples and has the advantage of being fully automated. In this study, we compare chemometric fingerprinting with a ribotyping fingerprinting method, in order to investigate the extent to which pyrogroups formed by PyMS analysis relate to genetic diversity, using polymerase chain reaction–restriction fragment length polymorphism–single-strand conformational polymorphism (PRS). A mixture of environmental strains of mycolic acid containing actinomycetes was used to mimic the selection of colonies from primary isolation plates. The congruence found between the clusters defined by the chemometric and molecular fingerprinting techniques was very high and demonstrated the effectiveness of PyMS as a rapid sorting and dereplicating procedure for putatively novel strains, criteria that are critical for biotechnological screens. Moreover, PyMS analysis revealed significant variation within pyrogroups that contained strains with the same genotypic (PRS) characteristics, thus emphasising its discriminatory capacity at the infraspecies level.

Survivability of bacteria ejected from icy surfaces after hypervelocity impact.

Both the Saturnian and Jovian systems contain satellites with icy surfaces. If life exists on any of these icy bodies (in putative subsurface oceans for example) then the possibility exists for transfer of life from icy body to icy body. This is an application of the idea of Panspermia, wherein life migrates naturally through space. A possible mechanism would be that life,here taken as bacteria, could become frozen in the icy surface ofone body. If a high-speed impact occurred on that surface, ejectacontaining the bacteria could be thrown into space. It could thenmigrate around the local region of space until it arrived at a second icy body in another high-speed impact. In this paper we consider some of the necessary steps for such a process to occur,concentrating on the ejection of ice bearing bacteria in the initial impact, and on what happens when bacteria laden projectiles hit an icy surface. Laboratory experiments using high-speed impacts with a light gas gun show that obtaining icy ejecta with viable bacterial loads is straightforward. In addition to demonstrating the viability of the bacteria carried on the ejecta, we have also measured the angular and size distribution of the ejecta produced in hypervelocity impacts on ice. We have however been unsuccessful at transferring viablebacteria to icy surfaces from bacteria laden projectilesimpacting at hypervelocities.

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study.

Gordonia namibiensis sp. nov., a novel nitrile metabolising actinomycete recovered from an African sand

A polyphasic approach was used to establish the taxonomic position of two actinomycetes isolated from a Namibian soil and shown to utilise nitrile compounds as growth substrates. The organisms, strains NAM-BN063AT and NAM-BN063B, had chemical and morphological properties consistent with their assignment to the genus Gordonia. Direct 165 rRNA sequencing studies confirmed the taxonomic position of the strains following the generation of phylogenetic trees using four different algorithms. The strains consistently formed a distinct phylogenetic line within the evolutionary radiation occupied by gordoniae and were most closely related to Gordonia rubropertincta DSM 43197T. DNA:DNA relatedness studies indicated that the two organisms belonged to a genomic species that was readily distinguished from G. rubropertincta. The unique phenotypic profile of the strains sharply separated them from representatives of all of the validly described species of Gordonia. The combination of genotypic and phenotypic data indicates that the two strains should be classified in the genus Gordonia as a new species. The name proposed for this taxon is Gordonia namibiensis, the type strain is NAM-BN063AT (= DSM 44568T = NCIMB 13780T).

Laboratory investigations of the survivability of bacteria in hypervelocity impacts

It is now well established that material naturally moves around the Solar System, even from planetary surface to planetary surface. Accordingly, the idea that life is distributed throughout space and did not necessarily originate on the Earth but migrated here from elsewhere (Panspermia) is increasingly deemed worthy of consideration. If life arrived at the Earth from space, its relative speed will typically be of order many km s-1, and the resulting collision with the Earth and its atmosphere will be in the hypervelocity regime. A mechanism for the bacteria to survive such an impact is required. Therefore a programme of hypervelocity impacts in the laboratory at (4.5 +/- 0.6) km s-1 was carried out using bacteria (Rhodococcus) laden projectiles. After impacts on a variety of target materials (rock, glass and metal) attempts were made to culture Rhodococcus from the surface of the resulting craters and also from the target material ejected during crater formation. Control shots with clean projectiles yielded no evidence for Rhodococcus growth from any crater surface or ejecta. When projectiles doped with Rhodococcus were used no impact crater surface yielded colonies of Rhodococcus. However, for four shots of bacteria into rock (two on chalk and two on granite) the ejecta was afterwards found to give colonies of Rhodococcus. This was not true for shots onto glass. In addition, shots into aerogel (density 96 kg m-3) were also carried out (two with clean projectiles and two with projectiles with Rhodococcus). This crudely simulated aero-capture in a planetary atmosphere. No evidence for Rhodococcus growth was found from the projectiles captured in the aerogel from any of the four shots.

Survivability of Bacteria in Hypervelocity Impacts on Ice

As part of the arrival stage of the Panspermia process, organisms must endure a hypervelocity impact either into the atmosphere or onto the surface of the destination planet. The impacts associated with this arrival stage are studied in this paper. To this end, the two-stage light gas gun at the University of Kent has been used to fire bacteria-laden projectiles, at velocities of approximately 5 km s−1, onto semi-solid nutrient medium, and solid and porous ice targets, representing planetary oceans and icy surfaces. The targets were then analysed to investigate whether the bacteria survived the impacts. It was found that bacteria can survive hypervelocity impacts at 5 km s−1, with a survival rate of 1 per 3.5 million using targets of nutrient gel. With ice targets no survival has been found yet with a limit on survival of less than 1 per 0.4 million.