A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716

Abstract

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICE_bph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICE_XTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICE_bph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICE_bph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICE_bph-salKF716 compared to other catabolic ICEs.

1. Introduction

Integrative and conjugative elements (ICEs) are mobile genetic elements of bacteria that are excised from the chromosome, transferred to other bacteria via conjugation, and reintegrated into the chromosome. They often carry cargo genes involved in antibiotic resistance, pathogenicity, heavy metal resistance, nitrogen fixation, or aromatic ring catabolism to impart beneficial traits to bacteria [1]. To date, a limited number of ICEs are known to carry cargo genes for the catabolism of aromatic polluting compounds, of which ICEclc is one of the best-characterized [2]. ICEclc contains cargo genes that encode the ortho-cleavage of chlorocatechol (clc genes) and aminophenol catabolism (amn genes). This element was originally identified in the 3-chlorobenzoic acid-degrading bacterium Pseudomonas knackmussii B13. Almost identical copies have been found on the chromosome of Burkholderia xenovorans LB400 (designated ICEclc-LB400) [3] and P. aeruginosa JB2 (ICEclc-JB2) [4]. The structure of ICEclc was compared with other conserved ICEs from five bacterial strains, but none of them contained a gene encoding the aromatic ring degradation pathway [5]. ICE_XTD from Azoarcus sp. CIB is another member of the ICEclc family, which is well characterized in terms of the function of cargo genes and the transferability by conjugation [6]. A notable feature of ICE_XTD is that it carries gene clusters for both aerobic and anaerobic degradation of xylene and toluene.

The bph genes, which are responsible for the co-metabolic degradation of polychlorinated biphenyls (PCBs), are widely distributed among both Gram-negative and Gram-positive bacteria [7]. Some bph genes are known to be horizontally transferred via mobile genetic elements. The first reported ICE carrying bph genes is Tn4371, found in the chromosome of the Gram-negative bacterium Cupriavidus oxalacticus A5 [8], with a total length of 61.8 kb. The chromosome of the Gram-negative bacterium Acidovorax sp. strain KKS102 contains ICE_KKS1024677, which belongs to the Tn4371 family [9]. ICE_KKS1024677 is known to transfer to a wide variety of bacteria across species and genera via a circular intermediate. Recently, we reported the entire genomes of ten PCB-degrading bacteria isolated from biphenyl-contaminated soil in Kitakyushu, Japan [10]. Among them, we detected ICEs carrying the bph gene from nine strains. ICE_bphKF708 from Cupriavidus basilensis KF708 and ICE_bphKF712 from Comamonas testosteroni KF712 are Tn4371 type ICEs, where ICE_bphKF708 is almost identical to ICE_KKS1024677. A 483-kb plasmid pKF715A carrying the bph gene and salicylic acid catabolic sal gene was detected from P. putida KF715, which could be transferred and integrated into the chromosome of P. putida AC30 or KT2440, and then maintained as ICE_bph-salKF715 [11]. Six Pseudomonas strains (P. abietaniphila KF701, P. aeruginosa KF702, P. putida KF703, P. furukawaii KF707 (formerly P. pseudoalcaligenes KF707), P. toyotomiensis KF710, and P. stutzeri KF716) carry an ICE_bph-sal element with sizes ranging from 117 kb to 130 kb, and integrate at the 3′ end of the tRNA-Gly(CCC) gene. It is obvious from their highly conserved sequences that ICE_bph-sals were generated via horizontal gene transfer. These ICEs carrying bph genes play an important role in the degradation of PCBs in the environment. Precise information on their structure and function will be important to understand the adaptation of their host strains to environmental niches and to design bioremediation processes using PCB-degrading bacteria. Here, we investigated the gene structure of ICE_bph-salKF716 in comparison with other catabolic ICEs and demonstrated that ICE_bph-salKF716 can be transferred via a circular intermediate form.

2. Materials and Methods

2.1. Bacterial Strains and Culture Conditions

Pseudomonas stutzeri KF716 (NBRC 110668), which has the ability to utilize biphenyl (Bph⁺) and salicylic acid (Sal⁺), was isolated from the soil in Kitakyushu, Japan [10]. P. aeruginosa PAO1(NBRC 106052), which has kanamycin resistance (Km^R), was obtained from the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC, Tokyo, Japan). For the growth of Pseudomonas strains, a basal salt medium containing (in grams per liter) K₂HPO₄, 4.3; KH₂PO₄, 3.4; (NH₄)₂SO₄, 2.0; MgCl₂·6H₂O, 0.34; MnCl₂·4H₂O, 0.001; FeSO₄·7H₂O, 0.0006; CaCl₂·2H₂O, 0.026; and Na₂MoO₄·2H₂O, 0.002 (pH 7.0) was used. The bacterial strains were grown by shaking at 120 rpm at 30 °C. For DNA isolation or freezing stock preparation, Luria–Bertani (LB) medium (Bacto Tryptone, 10 g; yeast extract, 5 g; and NaCl, 10 g/L, pH 7.0) was used.

2.2. Sequence Annotation and Computational Analysis

The sequence of ICE_bph-salKF716 was determined as previously reported [10,12]. The complete nucleotide sequence of ICE_bph-salKF716 has been deposited in DDBJ/ENA/GenBank under accession no. LC469614. The sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [13] and Rapid Annotations using Subsystems Technology (RAST) server v.2.0 [14]. The coding genes were identified using BLAST and BLASTX searches [15]. Sequence comparisons were performed using EasyFig v.2.1 [16], and a map was generated using the drawGeneArrows3 program (http://www.ige.tohoku.ac.jp/joho/labhome/tool.html, accessed on 28 November 2021). GC content and identity between genes were calculated using GENETYX version 15 (Genetyx Co. Ltd., Tokyo, Japan).

2.3. Detection of Target DNA by Polymerase Chain Reaction

Genomic DNA was extracted using a Genomic-tip 20/G (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Amplification of genes was performed in a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) and PCR conditions were performed in a 25 μL reaction mix containing 12.5 μL Gene RED PCR Mix Plus (Nippon Gene Co. Ltd., Toyama, Japan) at 94 °C for 5 min, followed by 30 cycles of 94 °C for 45 s, 62 °C for 45 s, and 72 °C for 60 s, with a final extension of 5 min at 72 °C. See Table S1 for primer sequences. PCR products were detected by agarose gel electrophoresis according to a standard procedure. The sequences of PCR products were determined using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI PRISM 3500 Genetic Analyzer.

2.4. Conjugal Transfer of ICE

Transfer of the Bph⁺ phenotype by conjugation into the recipient cells was carried out through filter mating. Donor and recipient cells were grown overnight in LB agar medium, and both cultures were suspended in 1 mL of LB broth. The cell suspensions (0.5 mL each) were mixed and placed on a nitrocellulose filter (0.45 μm, Merck Millipore, Bedford, MA, USA), and placed on an LB agar plate at 30 °C for 16 h. After incubation, the cells on the filter were suspended in sterilized saline, further diluted, and inoculated onto basal salt medium plates containing 30 μg of kanamycin, providing solid biphenyl in the inverted lid to select transconjugants. Conjugative transfer frequencies were calculated as the number of transconjugant cells per number of donor bacterial cells present in each mating.

3. Results and Discussion

3.1. Detection of ICE_bph-salKF716

A detailed analysis of the genome sequence of P. stutzeri KF716 revealed a genomic island of 117,300 bp (Figure 1), named ICE_bph-salKF716. ICE_bph-salKF716 is located at the 3’ end (attB site) of tRNAGly(CCC). The left end (attL) of ICE_bph-salKF716 is formed by 18 bp of tRNA-Gly (TTCCCTTCGCCCGCTCCA), and the right end (attR) is formed by a repetition of these 18 bp (Figure 2). We previously confirmed that this 18 bp direct repeat sequence is located on the border of the conserved ICE region and the non-conserved region of the chromosome [10]. The complete sequence of ICE_bph-salKF716 was submitted to the PGAP and RAST pipeline for annotation, and 112 ORFs were identified. Annotation was refined manually and compared with pKF715A [11], ICEclc [2], and ICE_XTD [6]. ICE_bph-salKF716 is a mobile genetic element that shares a core region with ICEclc and ICE_XTD, which have been shown to impart aromatic compound degradation genes to bacteria. The genetic maps of ICE_bph-salKF716, ICEclc, and ICE_XTD are shown in Figure 1, while their attL and attR are shown in Figure 2. The genes encoded by ICE_bph-salKF716 are listed in Table S2.

3.2. Core Region of ICE_bph-salKF716

The attL gene was found to be followed by a phage-related integrase (int) gene, which shares an identity with that of ICEclc or ICE_XTD carrying catabolic genes of aromatic compounds. ICE_bph-salKF716 contains core regions that are significantly similar to the core regions of ICEclc [2] and ICE_XTD [6] (Figure 1). The ORFs in the core region of ICE_bph-salKF716 were 55–83% identical to those of ICEclc, and 57–93% identical to those of ICE_XTD. The ORFs display the same genetic order in the ICE_bph-salKF716 element, and belong to the type IV secretion system (T4SS). Three factors have been recognized as important for DNA transfer by T4SS in Gram-negative and Gram-positive bacteria. These are: murein hydrolase [17], which is involved in controlled local degradation of the peptidoglycan, making space for the formation of a mating channel; the VirB4 ATPase, which provides energy for the translocation: and the VirD4 coupling protein, which links the DNA transfer intermediate to the mating channel [18]. In the ICE_bph-salKF716 element, we identified the virB4 gene (KF716ICE_480; the locus tags are listed in Table S2), virD4 gene (KF716ICE_650), and putative murein hydrolase (KF716ICE_670).

The parA and parB genes (KF716ICE_960, KF716ICE_940) encoding the replication partition proteins present 100 kb and 99 kb downstream of the attL site, respectively, were proposed to act as a stabilization system for the maintenance of mobile elements in the bacterial genomes [19]. Near parB, there is a gene encoding putative integrase regulator (KF716ICE_890) a homolog of InrR involved in the regulation of the expression of integrase on ICEclc [20]. Relaxase (traI, KF716ICE_370) which binds and nicks excised circular ICE at the origin of the transfer [21], TraG protein (traG, KF716ICE_410) [22], a component of the mating pair formation system, and pilin protein (pilL, KF716ICE_700) were also identified in the core region of ICE_bph-salKF716.

The homology of the integrase-encoding int gene between ICE_bph-sal and ICEclc was lower (66%) than that in the core region. The integration site of ICE_bph-sal was tRNA-Gly(CCC), whereas that of ICEclc was different in tRNA-Gly(GCC). In contrast, the homology of the int gene between ICE_XTD and ICE_bph-salKF716 was 70%; the integration site of ICE_XTD was tRNA-Gly(CCC), which was identical to that of ICE_bph-salKF716. The sequence of attL and attR at the end of ICE was five bases different between ICEclc and ICE_bph-salKF716, and one base difference between ICE_XTD and ICE_bph-salKF716 (Figure 2). The amino acid sequences of these ICE integrases may reflect different recognition of the integration site.

3.3. Variable Region of ICE_bph-salKF716

ICE_bph-salKF716 contains at least four variable regions (VR1–VR4) that are deficient in ICEclc and ICE_XTD (Figure 1). Variable region 1 (VR1) located near the attL site contains the biphenyl catabolic bph gene cluster with a total length of 11.2 kb, as well as a salicylate catabolic sal gene cluster with a total length of 11.5 kb. The bph gene cluster was found to be located just downstream of the int gene, followed by the sal gene cluster approximately 6 kb further downstream. The bph and sal genes encode for the degradation of biphenyl and salicylic acid to TCA cycle intermediates via an initial oxygenation step, followed by a meta-cleavage pathway. As shown in Figure 1, the bph gene cluster in VR1 shares 59–79% identity at the nucleotide sequence level with the toluene catabolic tod gene cluster located in the variable region of ICE_XTD in the opposite direction. The relationship between the bph gene of P. furukawaii KF707 and the tod gene of P. putida F1 has been reported in our previous paper [23]. The two variable regions (VR2 and VR3) are located at the midst of ICE_bph-salKF716. The GC content of these regions was lower than that of the other regions (Figure 3), and these ORFs (from KF716ICE_570 to KF716ICE_630 and from KF716ICE_710 to KF716ICE_740) were encoded in the opposite direction compared with ORFs in the surrounding core regions (Table S2), suggesting that these regions are inserted from the other genetic elements or chromosomes through horizontal gene transfer. VR2 contained ORFs coding for several hypothetical proteins with unknown functions, whereas VR3 is likely to be an insertion sequence since it includes an ORF identified as transposase (KF716ICE_720). The other variable region (VR4) located near the attR site contained the gene cluster coding for a putative ABC-type multi-drug efflux pump (from KF716ICE_1060 to KF716ICE_1110), with a total length of 6.6 kb that is deficient in ICEclc and ICE_XTD. The substrate of this transporter was not identified due to the lack of reliable homologous genes whose functions have been elucidated.

3.4. Comparison of ICE_bph-salKF716 and Other Putative ICEs

A search of the public database using BLAST revealed that an almost identical core region to that of ICE_bph-salKF716 was found in other putative ICEs. The identities were higher than those of ICEclc or ICE_XTD. Putative ICEs were identified from the genome sequences of P. putida B6-2 (accession number: NZ_CP015202) [24,25], P. alcaliphila JAB1 (CP016162) [26], P. stutzeri AN10 (NC_018028) [27], and P. stutzeri 2A20 (KT935509) [28]. They are flanked by directed repeat sequences corresponding to the attL and attR sites (Figure 2).

The putative ICE from P. putida B6-2 (tentatively designated as ICE_bph-salB6-2) has the closest relationship with ICE_bph-salKF716; it carries a bph-sal gene cluster that shares 91–100% identity with that of ICE_bph-salKF716, as well as highly conserved core regions (Figure 4). P. putida B6-2 is capable of degrading various polycyclic aromatic hydrocarbons [25]. Biphenyl (bph), salicylic acid (sal), and ferulic acid (fcs, ech), as well as downstream benzoic acid (ben) and protocatechuic acid (pca) catabolic gene clusters were identified on the genome sequence of P. putida B6-2, of which bph genes and sal genes are located in the ICE (Figure 4). The other putative ICE integrated in the genome of P. alcaliphila JAB1 (tentatively designated as ICE_bph-salJAB1) is the second-closest ICE to ICE_bph-salKF716; in this, the bph-sal gene cluster and benzoate catabolic bza gene cluster are included in the variable region (Figure 4). It is likely that inversion in the variable region, together with a part of the core region, occurred in the ICE_bph-salJAB1. In addition, sal:bza and bza:sal fusion gene clusters were found, in which parts of the sal genes and the bza genes were replaced with one another. This inversion at the sal-bza locus was also detected in ICE_bph-salKF702 of P. aeruginosa KF702, as described in our previous paper [10]. In addition to the well-characterized Tn4371 and ICE_KKS1024677, ICEs that carry the bph gene cluster included ICE_bph-salKF701, ICE_bph-salKF702, ICE_bph-salKF703, ICE_bph-salKF707, and ICE_bph-salKF710 from five biphenyl/PCB-degrading strains isolated from Kitakyushu, Japan [10]. ICE_bph-salKF716 shares a core region and bph-sal catabolic genes with these ICE_bph-sal elements, but lacks the bza gene, which encodes the benzoic acid degradation pathway. The comparison of the overall structure revealed that ICE_bph-salB6-2 from P. putida B6-2, and ICE_bph-salJAB1 from P. alcaliphila JAB1 are also members of the ‘ICE_bph-sal family’ (Figure 4). In particular, ICE_bph-salB6-2 is more closely related to ICE_bph-salKF716 as it lacks the bza gene. Since P. alcaliphila JAB1 was isolated in the Czech Republic, and P. stutzeri KF716 was isolated from Japan [10], it appears that ICE_bph-sals are globally distributed.

A BLAST search identified the other putative ICEs carrying a highly conserved core region with that of ICE_bph-salKF716 (Figure 5). The genome of P. stutzeri AN10 contained the putative ICE (tentatively designated ICE_nahAN10) carrying the naphthalene catabolic nah genes [29,30] that share more than 70–90% homology with the nah operon on plasmid NAH7 at the nucleotide sequence level. The aerobic degradation pathway of naphthalene consists of an upper pathway that transforms naphthalene to salicylic acid and pyruvic acid, and a lower pathway that transforms salicylic acid to TCA cycle intermediates. The nah lower operon coding for the lower pathway of ICE_nahAN10 shares 97–100% identity with the sal genes of ICE_bph-salKF716. In this context, it is likely that the nah upper operon is replaced by the bph genes in ICE_bph-salKF716, and, conversely, the bph genes are replaced by the nah upper operon in ICE_nahAN10. It has been confirmed that many putative mobile protein genes are present on ICE_bph-sals [10], which may act to replace the bph genes and the nah upper operon. The part of ICE_nahAN10, including the core region and genes coding for putative multi-drug efflux pumps, is perfectly identical (100%) to that of ICE_bph-salKF716, showing a very close relationship between the two ICEs. In fact, our previous paper described that the nah lower operon of P. stutzeri AN10 and the sal gene of P. furukawaii KF707 are highly conserved [31].

The putative ICE from P. stutzeri 2A20 (tentatively designated lCE_phe-xyl2A20) carried the tou genes coding for multicomponent toluene monooxygenases, phe genes coding for phenol meta-cleavage pathway, and xyl upper and lower operons coding for toluene-xylene catabolic genes [28] in the variable regions. The core regions of ICE_phe-xyl2A20, including partial phe genes, overlapped with the sal gene share 91–97% identity with those of ICE_bph-salKF716. A comparison of the variable regions of ICE_phe-xyl2A20 and ICE_bph-salKF716 strongly indicates that the substitution occurred between the biphenyl catabolic bph genes and toluene monooxygenase tou genes together with the part of the phe genes. Although ICE_nahAN10 and ICE_phe-xyl2A20 do not carry bph genes, the evolutionary relationship of ICE_bph-salKF716 with ICE_nahAN10 or ICE_phe-xyl2A20 was closer than that of ICEclc or ICE_XTD, as judged from the nucleotide sequence identity level. Their homologies of the core regions with ICE_bph-sal were higher (77–100% identity between ORFs) than those of ICEclc and ICE_XTD, indicating that these ICEs are more closely related to ICE_bph-salKF716. The identity of ICE_bph-salKF716 with ICE_nahAN10 or ICE_phe-xyl2A20 varies depending on the regions, where major parts including integrase (int) gene were highly conserved (90–100% identity), as shown in Figure 5. Although the variable regions among ICE_bph-salKF716, ICE_nahAN10, and ICE_phe-xyl2A20 are different, they commonly possess genes for aromatic compound meta-cleavage pathways. ICE_bph-salKF716, ICE_nahAN10, and ICE_phe-xyl2A20 are all ICEs found in P. stutzeri. This species is known to exhibit phenotypical diversity in the ecosystem and can adapt to the environmental niche [32], suggesting that it is particularly preferable as a host for this ICE subfamily.

3.5. Excision and Formation of Circular Intermediate Form of ICE_bph-salKF716

ICEs integrated into the chromosome can be excised from the chromosome to produce a circular form, and the host genome was repaired upon excision (Figure 6a). Attempts were made to detect the extrachromosomal circular form of ICE_bph-salKF716 from total DNA isolated from P. stutzeri KF716 grown on biphenyl. The amplicons corresponding to the attP site (R1 in Figure 6a) of excised circular forms of ICE_bph-salKF716, attB sites (R2) of excised closed forms of chromosome, and attL/attR sites (R3 and R4) of the integrated form were detected using PCR (Figure 6b). It has been reported that tRNA-Gly(CCC) locus is the insertion site of ICE_bph-salKF716 [10]. DNA sequences of these amplicons matched the expected sequences compared to the total genome sequence of P. stutzeri KF716 [12].

3.6. Transfer and Integration of ICE_bph-salKF716

To demonstrate the autonomous intercellular transfer of ICE_bph-salKF716, we performed mating experiments using P. stutzeri KF716 as a donor strain. P. aeruginosa PAO1 was used as the recipient strain because it carries Km^R phenotype, and forms a green colony, which enables it to be distinguished from the donor strain. The donor strain was mated with the recipient strain, and transconjugants were selected based on their Km^R and Bph⁺ phenotypes. We observed the appearance of transconjugants, P. aeruginosa PAO1 acquiring Bph⁺, Sal⁺ and Km^R phenotypes. The transfer frequencies (transconjugants per donor cell) ranged from 6.2 × 10⁻⁸ to 1.3 × 10⁻⁶, with 3.3 × 10⁻⁷ average in eight replicates. The transconjugants grew in a liquid medium containing biphenyl and salicylic acid as the sole source of carbon (data not shown). To confirm the identity of the transconjugant cells, PCR was performed with genomic DNA using primers 27F and 907R that amplify the 16S ribosomal DNA (16S rDNA), and the DNA sequence of the amplicon matched the 16S rDNA of the recipient strain. The amplicons corresponding to the attL/attR site (R6 and R7 in Figure 7a) of the integrated form of ICE_bph-salKF716 were detected from at least three PAO1 transconjugants. (Figure 7b). DNA sequencing these amplicons derived from the integrated form of ICE_bph-salKF716 in the transconjugants confirmed attB site at the 3′ end of tRNA-Gly(CCC) overlapping with sequence position 797,721 to 797,738 in genome sequence of P. putida PAO1 (accession number: NC_002516) [33]. The amplicons corresponding to the attP site (R1) of excised circular forms of ICE_bph-salKF716 and the attB site (R5) of excised closed form of chromosome were also detected from the transconjugants. DNA sequences of these amplicons matched the expected sequences compared to the attP site of ICE_bph-salKF716 or its insertion site on the P. aeriginosa PAO1 chromosome. At least two different sized amplicons were observed by electrophoresis (Figure 7b, Lane 3 and Lane 4) and by DNA sequencing when trying to obtain a fragment of R5 corresponding to the attB site or R6 corresponding to the attL site, respectively. This result indicates heterogeneity in the transconjugants, probably due to partial excision of ICE_bph-salKF716 from chromosome. It has been reported that the transfer efficiency of ICEclc is extremely high (1.4 × 10⁻²) [20], whereas that of ICE_KKS1024677 is extremely low (5.8 × 10⁻¹⁰) [9]. The transfer efficiency of ICE_bph-salKF716 was approximately 3.3 × 10⁻⁷ in the intermediate of ICEclc and ICE_KKS1024677 and comparable with that of ICE_XTD. The factors that govern the transfer efficiency of these ICEs remain to be elucidated, however many factors seem to involve the donor and recipient strains.

4. Conclusions

In this study, we investigated the structure and transfer of ICE_bph-salKF716, which is integrated into the chromosome of the biphenyl/PCB-degrading bacterium P. stutzeri KF716. Comparison with putative ICEs from other related Pseudomonas strains suggested the existence of a new ICEclc subfamily that shares nearly identical core regions. Since ICE_bph-salKF716 is the first member of this subfamily found, ICE_bph-salKF716 represents the ICEclc subfamily which is involved in the horizontal transfer of various catabolic genes among Pseudomonas strains. The results presented in this study will provide new insights into the evolution of ICEs in the process of adaptation to environmental niches, as well as a basis for designing bioremediation processes using PCB-degrading bacteria.