Jorge L. M. Rodrigues

Towards Environmental Systems Biology of Shewanella

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

Conversion of the Amazon rainforest to agriculture results in biotic homogenization of soil bacterial communities

The Amazon rainforest is the Earth’s largest reservoir of plant and animal diversity, and it has been subjected to especially high rates of land use change, primarily to cattle pasture. This conversion has had a strongly negative effect on biological diversity, reducing the number of plant and animal species and homogenizing communities. We report here that microbial biodiversity also responds strongly to conversion of the Amazon rainforest, but in a manner different from plants and animals. Local taxonomic and phylogenetic diversity of soil bacteria increases after conversion, but communities become more similar across space. This homogenization is driven by the loss of forest soil bacteria with restricted ranges (endemics) and results in a net loss of diversity. This study shows homogenization of microbial communities in response to human activities. Given that soil microbes represent the majority of biodiversity in terrestrial ecosystems and are intimately involved in ecosystem functions, we argue that microbial biodiversity loss should be taken into account when assessing the impact of land use change in tropical forests.

Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs)

Bacteria and functional genes associated with biphenyl (BP) degradation in the root zone of an Austrian pine (Pinus nigra L.) growing naturally in polychlorinated-BP (PCB)-contaminated soil were identified using stable isotope probing (SIP) integrated with comprehensive functional gene analyses. SIP revealed 75 different genera that derived carbon from 13C-BP, with Pseudonocardia, Kribella, Nocardiodes and Sphingomonas predominating carbon acquisition. Rhodococcus spp. were not detected with SIP, despite being the most abundant BP utilizers isolated from agar plates. Only one organism, an Arthrobacter spp., was detected as a BP utilizer by both cultivation and SIP methods. Time-course SIP analyses indicated that secondary carbon flow from BP-utilizing bacteria into other soil organisms may have occurred largely between 4 and 14 days incubation. Functional gene contents of the BP-utilizing metagenome (13C-DNA) were explored using the GeoChip, a functional gene array containing 6465 probes targeting aromatic degradative genes. The GeoChip detected 27 genes, including several associated with catabolism of BP, benzoate and a variety of aromatic ring hydroxylating dioygenase (ARHD) subunits. Genes associated with the β-ketoadipate pathway were also detected, suggesting a potential role for this plant aromatic catabolic pathway in PCB degradation. Further ARHD analyses using targeted polymerase chain reaction primers and sequence analyses revealed novel dioxygenase sequences in 13C-DNA, including several sequences that clustered distantly from all known ARHDs and others that resembled known Rhodococcus ARHDs. The findings improve our understanding of BP degradation and carbon flow in soil, reveal the extent of culture bias, and may benefit bioremediation research by facilitating the development of molecular tools to detect, quantify and monitor populations involved in degradative processes.

A composite bacteriophage alters colonization by an intestinal commensal bacterium

The mammalian intestine is home to a dense community of bacteria and its associated bacteriophage (phage). Virtually nothing is known about how phages impact the establishment and maintenance of resident bacterial communities in the intestine. Here, we examine the phages harbored by Enterococcus faecalis, a commensal of the human intestine. We show that E. faecalis strain V583 produces a composite phage (ϕV1/7) derived from two distinct chromosomally encoded prophage elements. One prophage, prophage 1 (ϕV1), encodes the structural genes necessary for phage particle production. Another prophage, prophage 7 (ϕV7), is required for phage infection of susceptible host bacteria. Production of ϕV1/7 is controlled, in part, by nutrient availability, because ϕV1/7 particle numbers are elevated by free amino acids in culture and during growth in the mouse intestine. ϕV1/7 confers an advantage to E. faecalis V583 during competition with other E. faecalis strains in vitro and in vivo. Thus, we propose that E. faecalis V583 uses phage particles to establish and maintain dominance of its intestinal niche in the presence of closely related competing strains. Our findings indicate that bacteriophages can impact the dynamics of bacterial colonization in the mammalian intestinal ecosystem.

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla(TEM-52) gene and two of them also harboured bla(TEM-1b). The bla(CTX-M-14) gene was identified in three isolates (in association with bla(TEM-1b) in one of them), and bla(CTX-M-32) was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions -42 (C-->T), -18 (G-->A), -1 (C-->T), and +58(C-->T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim-sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.

Seagulls of the Berlengas Natural Reserve of Portugal as Carriers of Fecal Escherichia coli Harboring CTX-M and TEM Extended-Spectrum Beta-Lactamases

ABSTRACT Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

Comparative systems biology across an evolutionary gradient within the Shewanella genus

To what extent genotypic differences translate to phenotypic variation remains a poorly understood issue of paramount importance for several cornerstone concepts of microbiology including the species definition. Here, we take advantage of the completed genomic sequences, expressed proteomic profiles, and physiological studies of 10 closely related Shewanella strains and species to provide quantitative insights into this issue. Our analyses revealed that, despite extensive horizontal gene transfer within these genomes, the genotypic and phenotypic similarities among the organisms were generally predictable from their evolutionary relatedness. The power of the predictions depended on the degree of ecological specialization of the organisms evaluated. Using the gradient of evolutionary relatedness formed by these genomes, we were able to partly isolate the effect of ecology from that of evolutionary divergence and to rank the different cellular functions in terms of their rates of evolution. Our ranking also revealed that whole-cell protein expression differences among these organisms, when the organisms were grown under identical conditions, were relatively larger than differences at the genome level, suggesting that similarity in gene regulation and expression should constitute another important parameter for (new) species description. Collectively, our results provide important new information toward beginning a systems-level understanding of bacterial species and genera.

DNA-Stable Isotope Probing Integrated with Metagenomics for Retrieval of Biphenyl Dioxygenase Genes from Polychlorinated Biphenyl-Contaminated River Sediment

ABSTRACT Stable isotope probing with [13C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [13C]DNA after a 14-day incubation with [13C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the [13C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the [13C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits bphAE, while the rest of the clones sequence was similar to that of an unknown member of the Gammaproteobacteria. A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.

Mechanisms of Antibiotic Resistance in Escherichia coli Isolates Recovered from Wild Animals

Seventy-two fecal samples obtained from wild animals in Portugal were sampled on Levine agar plates (non-supplemented with antibiotics), and Escherichia coli isolates were recovered from 56 of them (78%), obtaining a total of 112 E. coli isolates (two per sample). Susceptibility to 16 antibiotics was studied in these isolates, and the following percentages of resistance were obtained: tetracycline, streptomycin, ampicillin, and trimethoprim-sulfamethoxazole (SXT) (range 19-35%); nalidixic acid (14%); ciprofloxacin (9%); amoxicillin-clavulanic acid, gentamicin, tobramycin, and chloramphenicol (range 4.5-7%); cefotaxime, and aztreonam (1.8%); ceftazidime (0.9%); and amikacin, cefoxitin, and imipenem (0%). A bla(TEM) gene was found in 22 of the 25 ampicillin-resistant isolates, and the gene encoding CTX-M-14 beta-lactamase was identified in the two cefotaxime-resistant isolates (recovered from a common kestrel and a sparrowhawk), associated with bla(TEM-52) gene in one of them. Other resistance genes detected were as follows: aac(3)-II or aac(3)-IV genes in all gentamicin-resistant isolates; aadA1 or aadA2 in 22 of 25 streptomycin-resistant isolates; tet(A) and/or tet(B) in all 39 tetracycline-resistant isolates; and sul1 and/or sul2 and/or sul3 genes in all 21 SXT-resistant isolates. Two amino acid changes in GyrA protein (Ser83Leu + Asp87Asn) and one change in ParC protein (Ser80Ile) were identified in all 10 ciprofloxacin-resistant isolates of our series. The intestinal tract of wild animals is a reservoir of antibiotic resistance genes, especially for ampicillin, tetracycline, streptomycin, and SXT, and it is also remarkable that multiresistant E. coli isolates are detected in some of the tested animals.

Degradation of Aroclor 1242 Dechlorination Products in Sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. Strain RHA1(fcb)

ABSTRACT Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (104 or 106 cells g−1 sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.