Amy K. Klimowicz

Culturing captures members of the soil rare biosphere

The ecological significance of rare microorganisms within microbial communities remains an important, unanswered question. Microorganisms of extremely low abundance (the ‘rare biosphere’) are believed to be largely inaccessible and unknown. To understand the structure of complex environmental microbial communities, including the representation of rare and prevalent community members, we coupled traditional cultivation with pyrosequencing. We compared cultured and uncultured bacterial members of the same agricultural soil, including eight locations within one apple orchard and four time points. Our analysis revealed that soil bacteria captured by culturing were in very low abundance or absent in the culture-independent community, demonstrating unexpected accessibility of the rare biosphere by culturing.

Genetics of Zwittermicin A Production by Bacillus cereus

ABSTRACT Zwittermicin A represents a new chemical class of antibiotic and has diverse biological activities, including suppression of oomycete diseases of plants and potentiation of the insecticidal activity of Bacillus thuringiensis. To identify genes involved in zwittermicin A production, we generated 4,800 transposon mutants of B. cereus UW101C and screened them for zwittermicin A accumulation. Nine mutants did not produce detectable zwittermicin A, and one mutant produced eightfold more than the parent strain. The DNA flanking the transposon insertions in six of the nine nonproducing mutants contains significant sequence similarity to genes involved in peptide and polyketide antibiotic biosynthesis. The mutant that overproduced zwittermicin A contained a transposon insertion immediately upstream from a gene that encodes a deduced protein that is a member of the MarR family of transcriptional regulators. Three genes identified by the mutant analysis mapped to a region that was previously shown to carry the zwittermicin A self-resistance gene, zmaR, and a biosynthetic gene (E. A. Stohl, J. L. Milner, and J. Handelsman, Gene 237:403-411, 1999). Further sequencing of this region revealed genes proposed to encode zwittermicin A precursor biosynthetic enzymes, in particular, those involved in the formation of the aminomalonyl- and hydroxymalonyl-acyl carrier protein intermediates. Additionally, nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) homologs are present, suggesting that zwittermicin A is synthesized by a mixed NRPS/PKS pathway.

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units

Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.

Use of a Promoter Trap to Identify Bacillus cereus Genes Regulated by Tomato Seed Exudate and a Rhizosphere Resident Pseudomonas aureofaciens

ABSTRACT The goal of this study was to identify genes in Bacillus cereus, a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident, Pseudomonas aureofaciens. We constructed a B. cereus chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene, gfpmut3a. The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of P. aureofaciens strain 30-84. We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by P. aureofaciens culture supernatant. A clone chosen for further study contained an open reading frame, designated lipA, that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in B. subtilis. Expression of gusA under control of the lipA promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids. When the wild type and a 10-fold excess of a lipA mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the lipA mutant was unaffected. This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of lipA, a plant-regulated gene in B. cereus.

Psychrotrophic Strain of Janthinobacterium lividum from a Cold Alaskan Soil Produces Prodigiosin

We have explored the microbial community in a nonpermafrost, cold Alaskan soil using both culture-based and culture-independent approaches. In the present study, we cultured >1000 bacterial isolates from this soil and characterized the collection of isolates phylogenetically and functionally. A screen for antibiosis identified an atypical, red-pigmented strain of Janthinobacterium lividum (strain BR01) that produced prodigiosin when grown at cool temperatures as well as strains (e.g., strain BP01) that are more typical of J. lividium, which produce a purple pigment, violacein. Both purple- and red-pigmented strains exhibited high levels of resistance to beta-lactam antibiotics. The prodigiosin pathway cloned from J. lividium BR01 was expressed in the heterologous host, Escherichia coli, and the responsible gene cluster differs from that of a well-studied prodigiosin producer, Serratia sp. J. lividum BR01 is the first example of a prodigiosin-producer among the beta-Proteobacteria. The results show that characterization of cultured organisms from previously unexplored environments can expand the current portrait of the microbial world.

Peptidoglycan from Bacillus cereus mediates commensalism with rhizosphere bacteria from the Cytophaga-Flavobacterium group.

ABSTRACT Previous research in our laboratory revealed that the introduction of Bacillus cereus UW85 can increase the populations of bacteria from the Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman, Ecology 74:840-854, 1993). In the present study, we determined the frequency at which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere and the mechanism by which B. cereus stimulates the growth of CF rhizosphere strains in root exudate media. In three consecutive years of sampling, CF strains predominated among coisolates obtained with B. cereus isolates from field-grown soybean roots. In root exudate media, the presence of B. cereus was required for CF coisolate strains to reach high population density. However, rhizosphere isolates from the phylum Proteobacteria grew equally well in the presence and absence of B. cereus, and the presence of CF coisolates did not affect the growth of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture supernatant from B. cereus grown in root exudate media did not. These results suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.

Streptomycin Application Has No Detectable Effect on Bacterial Community Structure in Apple Orchard Soil

ABSTRACT Streptomycin is commonly used to control fire blight disease on apple trees. Although the practice has incited controversy, little is known about its nontarget effects in the environment. We investigated the impact of aerial application of streptomycin on nontarget bacterial communities in soil beneath streptomycin-treated and untreated trees in a commercial apple orchard. Soil samples were collected in two consecutive years at 4 or 10 days before spraying streptomycin and 8 or 9 days after the final spray. Three sources of microbial DNA were profiled using tag-pyrosequencing of 16S rRNA genes: uncultured bacteria from the soil (culture independent) and bacteria cultured on unamended or streptomycin-amended (15 μg/ml) media. Multivariate tests for differences in community structure, Shannon diversity, and Pielous evenness test results showed no evidence of community response to streptomycin. The results indicate that use of streptomycin for disease management has minimal, if any, immediate effect on apple orchard soil bacterial communities. This study contributes to the profile of an agroecosystem in which antibiotic use for disease prevention appears to have minimal consequences for nontarget bacteria.

A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal

Bacillus thuringiensis is the leading biopesticide used to control insect pests worldwide. Although they have a long record of safe use, under certain conditions commercial strains of B. thuringiensis have the ability to produce numerous putative enterotoxins that have been associated with food poisoning attributed to Bacillus cereus. Therefore, we designed a strategy to delete the genes encoding these toxins. B. thuringiensis strain VBTS 2477 contained genes encoding NHE, CytK-2 and three homologues of haemolysin BL (HBL, HBL(a1) and HBL(a2)). This is the first report, to our knowledge, of a strain of B. cereus or B. thuringiensis containing three sets of hbl operons. The genes encoding HBL(a1) and HBL(a2) were 96-97 % identical to each other and 76-84 % identical to those encoding HBL. The hbl(a2) operon was detected by PCR amplification only after hbl(a1) was deleted. We used sequential gene replacement to replace the wild-type copies of the NHE and three HBL operons with copies that contained internal deletions that span the three genes in each operon. The insecticidal activity of the quadruple-enterotoxin-deficient mutant was similar to that of the wild-type strain against larvae of Trichoplusia ni, Spodoptera exigua and Plutella xylostella. This demonstrates that the genes for enterotoxins can be deleted, eliminating the possibility of enterotoxin production without compromising the insecticidal efficacy of a strain of B. thuringiensis.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady‐state levels of two T4SS structural proteins were affected by a homolog of tail‐specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem‐loop structures contained within the 5′ untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post‐translational mechanisms controlling T4SS gene expression and DNA secretion.