Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments.

IMPORTANCE: PP biodegradation has not been clearly shown (it has been uncertain whether the PP structure is actually biodegraded or not). This is the first report on the obvious biodegradation of PP. At the same time, this study shows that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Moreover, PP biodegradation has been investigated by using solid PP as the sole carbon source. However, this study shows that PP would not be used as a sole carbon and energy source. Our data thus provide very important and key knowledge for PP bioremediation.

Lolliginicoccus levis gen. nov., sp. nov., a novel bacterium isolated from the brain of the Chiroteuthis picteti squid, and reclassification of two Hoyosella species as Lolliginicoccus suaedae comb. nov. and Lolliginicoccus lacisalsi comb. nov.

A Gram-stain-positive, non-motile, mesophilic, aerobic, coccus-shaped bacterium, designated strain Y7R2T, was isolated from the brain of a Chiroteuthis picteti squid living in mesopelagic water near Muroto, Kochi, Japan. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain was most closely related to the genus Hoyosella (96.1 % similarity to the type strain of the type species Hoyosella altamirensis) and formed a separate distinct cluster in a stable, deep-branching lineage with the type strains of Hoyosella suaedae and Hoyosella lacisalsi (98.7-99.5% similarities). The major fatty acids (>10 %) of strain Y7R2T were C17 : 1 ω8c, C15 : 0, C16 : 1 ω6c/C16 : 1 ω7c and C16 : 0, and the isoprenoid quinones were menaquinone-7 (57.8 %) and menaquinone-8 (42.2 %). The principal polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol, and the DNA G+C content was 68.0 %. These chemotaxonomic features, with the exception of the fatty acid composition, were similar to those of the phylogenetically clustered species (H. suaedae and H. lacisalsi) but different from those of core Hoyosella species (including H. altamirensis). These results suggested that Y7R2T, H. suaedae and H. lacisalsi strains should be assigned to a novel genus. Furthermore, strain Y7R2T showed low average nucleotide identity values (88.0-88.2 %) and low digital DNA-DNA hybridization values (34.3-34.7 %) to the type strains of H. suaedae and H. lacisalsi. These data indicated that strain Y7R2T should be assigned to a novel genus and species, for which the name Lolliginicoccus levis gen. nov., sp. nov. is proposed. The type strain is Y7R2T (=NBRC 114883T=KCTC 49749T). Accordingly, reclassification of H. suaedae and H. lacisalsi as Lolliginicoccus suaedae comb. nov. (type species) and Lolliginicoccus lacisalsi comb. nov. is also proposed.

Aurantiacicella marina gen. nov., sp. nov., a myxol-producing bacterium from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2A-8T, was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced myxol as a major carotenoid. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and was related most closely to the genus Aquimarina (91.0-94.4 % 16S rRNA gene sequence similarity to the type strains of species of this genus). The DNA G+C content was 35 mol%. The major fatty acids were iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified aminolipid and five unidentified lipids. Menaquinone 6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-8T represents a novel genus and species, for which the name Aurantiacicella marina gen. nov., sp. nov. is proposed. The type strain of Aurantiacicella marina is 2A-8T ( = NBRC 111187T = KCTC 42676T).

Flavicella marina gen. nov., sp. nov., a carotenoid-producing bacterium from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped or spherical bacterium, strain 2A-7(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain produced a pigment(s), the absorption spectrum of which closely resembled that of ß-carotene. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Flavobacteriaceae and clustered distantly with the type strains of species of the genus Lutibacter (up to 93.9 % similarity). The DNA G+C content was 34.1 mol%. The major fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. Menaquinone 6 was detected as the sole isoprenoid quinone. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 2A-7(T) represents a novel genus and species, for which the name Flavicella marina gen. nov., sp. nov. is proposed. The type strain of Flavicella marina is 2A-7(T) ( = NBRC 110145(T) = KCTC 42197(T)).

Perspicuibacter marinus gen. nov., sp. nov., a semi-transparent bacterium isolated from surface seawater, and description of Arenicellaceae fam. nov. and Arenicellales ord. nov.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, strain 2-9(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain was transparent on 1/5 strength marine broth plate but became easily visible when the plate was supplemented with pyruvate. Phylogenetic analyses based on the 16S rRNA gene sequence showed that the strain fell within the class Gammaproteobacteria and was most closely related to the genus Arenicella (92.7-93.0 % 16S rRNA gene sequence similarities to type strains of species of this genus) of an unclassified order within this class. The DNA G+C content of strain 2-9(T) was 41.7 mol%. The major fatty acids were C18 : 1ω7c (37.6 %), C16 : 1ω7c and/or iso-C15 : 0 2-OH (summed feature 3; 19.1 %), C18 : 0 (10.8 %), C16 : 0 (10.2 %) and an unidentified fatty acid with an equivalent chain-length value of 11.799 (9.5 %). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. Ubiquinone-8 (Q-8) was detected as the sole isoprenoid quinone. From these taxonomic data, it is proposed that strain 2-9(T) represents a novel species of a new genus, Perspicuibacter marinus gen. nov., sp. nov. The type strain of the type species is 2-9(T) ( = NBRC 110144(T) = KCTC 42196(T)). A new family, Arenicellaceae fam. nov. (type genus Arenicella), and order, Arenicellales ord. nov., of the class Gammaproteobacteria are proposed to accommodate the novel taxon.

Amylibacter marinus gen. nov., sp. nov., isolated from surface seawater.

A Gram-stain-negative, non-motile, mesophilic, aerobic, rod-shaped bacterium, designated strain 2-3(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. This strain grew well with starch. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Rhodobacteraceae and that the strain was related most closely to the genus Pacificibacter (94.0 % sequence similarity to the type strain). The DNA G+C content was 52.4 mol%. The major fatty acids were C18 : 1ω7c, C14 : 0 and C16 : 0. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified lipid, one unidentified aminolipid and one unidentified phospholipid. The major isoprenoid quinone was Q-10. Strain 2-3(T) did not grow at 4 or 35 °C, while the type strain of the type species of the genus Pacificibacter grows at both temperatures. From the taxonomic data obtained in this study, it is proposed that strain 2-3(T) be placed into a novel genus and species named Amylibacter marinus gen. nov., sp. nov. in the family Rhodobacteraceae. The type strain of Amylibacter marinus is 2-3(T) ( = NBRC 110140(T) = LMG 28364(T)).

Temperatibacter marinus gen. nov., sp. nov., a mesophilic bacterium isolated from surface seawater and description of Temperatibacteraceae fam. nov. in the class Alphaproteobacteria.

A Gram-stain-negative, motile, mesophilic, aerobic, rod-shaped bacterium, strain 5-11(T), was isolated from surface seawater at Muroto city, Kochi prefecture, Japan. The strain exhibited a narrow growth temperature range of 20-30 °C. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the order Kordiimonadales in the class Alphaproteobacteria and was related most closely to the genus Kordiimonas (up to 91.2 % similarity to the type strains of species of the genus) but branched deeply from species of Kordiimonas. The major fatty acids were iso-C17 : 1ω9c, iso-C15 : 0, and C16 : 1ω7c and/or iso-C15 : 0 2-OH. Ubiquinone-10 (Q-10) was detected as the sole isoprenoid quinone. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminolipid. Although strains of Kordiimonas have been shown to contain unidentified glycolipids, they were not detected from strain 5-11(T). The DNA G+C content of strain 5-11(T) was 44.3 mol%, a value that was lower than those of strains of Kordiimonas (50-58 mol%) and was relatively low for the members of the class Alphaproteobacteria. On the basis of phenotypic, genotypic and chemotaxonomic data, it is proposed that strain 5-11(T) represents a novel species of a new genus, Temperatibacter marinus gen. nov., sp. nov., within a new family Temperatibacteraceae fam. nov. The type strain of Temperatibacter marinus is 5-11(T) ( = NBRC 110045(T) = LMG 28278(T)).

Agaribacter marinus gen. nov., sp. nov., an agar-degrading bacterium from surface seawater.

A Gram-stain-negative, motile, mesophilic, aerobic, rod-shaped bacterium, strain 8-8(T), was isolated from surface seawater at Muroto, Kochi, Japan. The strain exhibited agar-degrading activity. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain fell within the family Alteromonadaceae and clustered distantly with members of the genus Glaciecola (≤ 94.0% similarity). The DNA G+C content was 41.8 mol%. The major fatty acids were C16 : 1ω7c and/or iso-C15 : 0 2-OH, C16 : 0 and C18 : 1ω7c and the major hydroxy fatty acid was C12 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unidentified polar lipid; lysophosphatidylethanolamine and unidentified polar lipids were found as minor components. The major quinone was Q-8. On the basis of phenotypic, genotypic and chemotaxonomic data, strain 8-8(T) represents a novel species of a new genus, for which the name Agaribacter marinus gen. nov., sp. nov. is proposed. The type strain of Agaribacter marinus is 8-8(T) ( = NBRC 110023(T) = LMG 28167(T)).

Oleibacter marinus gen. nov., sp. nov., a bacterium that degrades petroleum aliphatic hydrocarbons in a tropical marine environment.

Three Gram-negative, motile, mesophilic, aerobic, rod-shaped bacterial strains, designated 2O1(T), 1O14 and 1O18, were isolated from Indonesian seawater after enrichment with crude oil and a continuous supply of supplemented seawater. The strains exhibited high n-alkane-degrading activity, which indicated that the strains were important degraders of petroleum aliphatic hydrocarbons in tropical marine environments. Phylogenetic analyses based on 16S rRNA gene sequences of members of the Gammaproteobacteria showed that the isolates formed a coherent and distinct cluster in a stable lineage containing Oceanobacter kriegii IFO 15467(T) (96.4-96.5 % 16S rRNA gene sequence similarity) and Thalassolituus oleivorans MIL-1(T). DNA G +C content was 53.0-53.1 mol%. The major fatty acids were C(16 : 0), C(16 : 1)ω7 and C(18 : 1)ω9 and the hydroxy fatty acids were C(12 : 0) 3-OH and C(10 : 0) 3-OH. The polar lipids were phosphatidylglycerol, a ninhydrin-positive phospholipid(s) and glycolipids. The major quinone was Q-9 (97-99 %), which distinguished the isolates from Oceanobacter kriegii NBRC 15467(T) (Q-8; 91 %). On the basis of phenotypic, genotypic and chemotaxonomic data, including DNA-DNA hybridization, the isolates represent a novel genus and species, for which the name Oleibacter marinus gen. nov., sp. nov. is proposed. The type strain of Oleibacter marinus is 2O1(T) (=NBRC 105760(T) =BTCC B-675(T)).

The Bacterial Community in the Western Region of North Pacific Intermediate Water.

The bacterial composition in North Pacific Intermediate Water (NPIW) was investigated in three different years and compared with that in other seawaters around Japan. The results indicated that bacterial composition was surprisingly stable at the same point in a mesopelagic water mass throughout the years and supported previous physicochemical observations that NPIW is distributed to Kumejima, Japan.

A gene cluster for fatty alcohol synthesis from a Reinekea-related bacterium that accumulates fatty alcohols.

This study reports on a marine bacterium that accumulates fatty alcohols (C14,16,18 ) at more than 1% (w/w) of the dry cell weight. This unique bacterium, designated as strain 1-4, is related to the genus Reinekea. A novel gene cluster for fatty alcohol synthesis, phsAB, is identified from strain 1-4. The phsA product shows significant homology to fatty acyl-CoA reductase (51% identity), whereas the phsB product shows very low homology to lipases. Interestingly, phsA alone causes Escherichia coli to accumulate fatty alcohols at 19% (w/w) of the dry cell weight. Moreover, the phsA-containing E. coli accumulate more fatty alcohols (24%) and grow faster after phsB is introduced, indicating that phsAB could greatly assist the mass production of fatty alcohols.

Genome Sequence of the Psychrophilic Bacterium Tenacibaculum ovolyticum Strain da5A-8 Isolated from Deep Seawater.

Some bacterial species of the genus Tenacibaculum, including Tenacibaculum ovolyticum, have been known as fish pathogens in the sea. So far, the only published genome sequence for this genus is for Tenacibaculum dicentrarchi, which could also be a fish pathogen. Strain da5A-8, showing 100% identity to the 16S rRNA gene sequence of T. ovolyticum DSM 18103(T), was isolated from seawater at a depth of 344 m in Kochi, Japan, and grew optimally at 10 to 20°C. The genome sequence of strain da5A-8 revealed the possible virulence genes commonly observed in the genus Tenacibaculum.

1-Hydroxy monocyclic carotenoid 3,4-dehydrogenase from a marine bacterium that produces myxol.

A crtD (1-HO carotenoid 3,4-dehydrogenase gene) homolog from marine bacterium strain P99-3 included in the gene cluster for the biosynthesis of myxol (3',4'-didehydro-1',2'-dihydro-beta, psi-carotene-3,1',2'-triol) was functionally identified. The P99-3 CrtD was phylogenetically distant from the other CrtDs. A catalytic feature was its high activity for the monocyclic carotenoid conversion: 1'-HO-torulene (3',4'-didehydro-1',2'-dihydro- beta, psi-caroten-1'-ol) was prominently formed from 1'-HO-gamma-carotene (1',2'-dihydro-beta, psi-caroten-1'-ol) in Escherichia coli with P99-3 CrtD, indicating that this enzyme has been highly adapted to myxol biosynthesis. This unique type of crtD is a valuable tool for obtaining 1'-HO-3',4'-didehydro monocyclic carotenoids in a heterologous carotenoid production system.

Structural and functional analysis of a lycopene beta-monocyclase gene isolated from a unique marine bacterium that produces myxol.

A gene coding for lycopene beta-monocyclase, which metabolizes lycopene (psi,psi-carotene) to gamma-carotene (beta,psi-carotene), was isolated for the first time from a unique marine bacterium strain P99-3 that produces myxol (a gamma-carotene derivative). This lycopene beta-monocyclase gene (designated crtYm) was included in the gene cluster which contained carotenoid biosynthetic gene (crtI, crtB, crtZ, crtY, and crtA) homologs. CrtYm, the CrtY homolog, metabolized lycopene to gamma-carotene, which was confirmed by deletion/expression analysis of the crtYm and by subsequent analysis of the metabolites from lycopene based on the retention times on high-performance liquid chromatography, UV-visible absorption spectra, and mass spectrometry.

Specialized Hydrocarbonoclastic Bacteria Prevailing in Seawater around a Port in the Strait of Malacca.

Major degraders of petroleum hydrocarbons in tropical seas have been indicated only by laboratory culturing and never through observing the bacterial community structure in actual environments. To demonstrate the major degraders of petroleum hydrocarbons spilt in actual tropical seas, indigenous bacterial community in seawater at Sentosa (close to a port) and East Coast Park (far from a port) in Singapore was analyzed. Bacterial species was more diverse at Sentosa than at the Park, and the composition was different: γ-Proteobacteria (57.3%) dominated at Sentosa, while they did not at the Park. Specialized hydrocarbonoclastic bacteria (SHCB), which use limited carbon sources with a preference for petroleum hydrocarbons, were found as abundant species at Sentosa, indicating petroleum contamination. On the other hand, SHCB were not the abundant species at the Park. The abundant species of SHCB at Sentosa were Oleibacter marinus and Alcanivorax species (strain 2A75 type), which have previously been indicated by laboratory culturing as important petroleum-aliphatic-hydrocarbon degraders in tropical seas. Together with the fact that SHCB have been identified as major degraders of petroleum hydrocarbons in marine environments, these results demonstrate that the O. marinus and Alcanivorax species (strain 2A75 type) would be major degraders of petroleum aliphatic hydrocarbons spilt in actual tropical seas.

The potential of Cycloclasticus and Altererythrobacter strains for use in bioremediation of petroleum-aromatic-contaminated tropical marine environments.

Cycloclasticus sp. A5, which has been suggested to be a major degrader of petroleum aromatics spilled in temperate seas, showed higher degrading activities for petroleum aromatics, at both 25 degrees C and tropical sea temperature 30 degrees C, than the novel aromatic-degrading isolates, related to Altererythrobacter epoxidivorans (97.5% similarity in the almost full-length 16S rRNA gene sequence) and Rhodovulum iodosum (96.3% similarity), obtained after enrichment on crude oil in a continuous supply of Indonesian seawater. Cycloclasticus A5 degraded petroleum aromatics at a similar rate or faster at 30 degrees C as compared to 25 degrees C, but its growth on acetate was severely inhibited at 30 degrees C. These results suggest that, although their abundance would be low in tropical seas not contaminated with aromatics, the Cycloclasticus strains could be major degraders of petroleum aromatics spilled in tropical seas. The 16S rRNA gene of the Cycloclasticus strains has been identified from Indonesian seawater, and the gene fragments showed 96.7-96.8% similarities to that of Cycloclasticus A5. Introducing Cycloclasticus A5 may be an ecologically advantageous bioremediation strategy for petroleum-aromatic-contaminated tropical seas because strain A5 would disappear at 30 degrees C after complete consumption of the aromatics. Altererythrobacter and Rhodovulum-related isolates grew well on pyruvate in 10% strength marine broth at 30 degrees C whereas Cycloclasticus A5 did not grow well on acetate in the broth at 30 degrees C. These growth results, along with its petroleum-aromatic-degrading activity, suggest that the Altererythrobacter isolate could be an important petroleum-aromatic degrader in and around nutrient-rich tropical marine environments.

Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment.

Petroleum-hydrocarbon-degrading bacteria were obtained after enrichment on crude oil (as a 'chocolate mousse') in a continuous supply of Indonesian seawater amended with nitrogen, phosphorus and iron nutrients. They were related to Alcanivorax and Marinobacter strains, which are ubiquitous petroleum-hydrocarbon-degrading bacteria in marine environments, and to Oceanobacter kriegii (96.4-96.5 % similarities in almost full-length 16S rRNA gene sequences). The Oceanobacter-related bacteria showed high n-alkane-degrading activity, comparable to that of Alcanivorax borkumensis strain SK2. On the other hand, Alcanivorax strains exhibited high activity for branched-alkane degradation and thus could be key bacteria for branched-alkane biodegradation in tropical seas. Oceanobacter-related bacteria became most dominant in microcosms that simulated a crude oil spill event with Indonesian seawater. The dominance was observed in microcosms that were unamended or amended with fertilizer, suggesting that the Oceanobacter-related strains could become dominant in the natural tropical marine environment after an accidental oil spill, and would continue to dominate in the environment after biostimulation. These results suggest that Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in the tropical sea.

Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB.

Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.

Stable augmentation of activated sludge with foreign catabolic genes harboured by an indigenous dominant bacterium.

Comamonas sp. rN7 is a phenol-degrading bacterium that represents the dominant catabolic population in activated sludge. The present study examined the utility of this bacterium for establishing foreign catabolic genes in phenol-digesting activated sludge. The phc genes coding for phenol hydroxylase and its transcriptional regulators of C. testosteroni R5 were integrated into the chromosome of strain rN7. The specific phenol-oxygenating activity of a resultant transformant designated rN7(R503) was three times higher than the activity of strain rN7, and the phc genes were stably inherited by rN7(R503) grown in a non-selective laboratory medium. Inoculation of phenol-acclimatized activated sludge with rN7(R503) resulted in a high phenol-oxygenating activity and improved resistance to phenol-shock loading compared to sludge inoculated with either no cells, rN7 or R5. Quantitative competitive polymerase chain reaction (PCR) showed that the phc genes were retained in the rN7(R503)-inoculated sludge at a density of more than 108 copies per ml of mixed liquor for more than 35 days, whereas those in the R5-inoculated sludge disappeared rapidly. No transfer of the phc genes to other indigenous populations was apparent in the rN7(R503)-harbouring sludge. From these results, we concluded that the phenol treatment of the activated sludge was enhanced by the phc genes harboured by the rN7(R503) population. This study suggests a possible bioaugmentation strategy for stably utilizing foreign catabolic genes in natural ecosystems.

An AraC/XylS family member at a high level in a hierarchy of regulators for phenol-metabolizing enzymes in Comamonas testosteroni R5.

Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.