Isolation, sequencing and expression of the gene encoding a major protein from the backteriophage associated with Bartonella henselae.

The gene encoding a 31-kDa major protein (Pap31) associated with the bacteriophage harbored in Bartonella henselae was cloned and sequenced. Analysis of the resulting sequence revealed an open reading frame of 837 nucleotides coding for a protein of 279 amino acids. pap31 was then subcloned downstream of the lacZ promoter in pUC19. pap31 was amplified by polymerase chain reaction, and the linear amplicon was used as template for in-vitro transcription and translation. A protein with an apparent molecular mass of approximately 31 kDa was synthesized from this reaction. Upon analysis of the deduced aa sequence, a potential signal sequence and a consensus signal peptidase cleavage site were identified, indicative that Pap31 is modified posttranslationally, and the mature protein may be targeted to the host membrane.

Analysis of 36-kilodalton protein (PapA) associated with the bacteriophage particle of Bartonella henselae.

A library of Bartonella henselae DNA was screened with antibody raised to the bacteriophage particle associated with this organism. A clone was isolated that expresses a 36-kD protein (termed PapA for particle-associated protein) when examined by immunoblot analysis using antibody raised to the particle. Southern blot hybridization indicates that the gene is present on the bacterial chromosome and packaged into the 14-kb particle-associated DNA. A papA-specific probe hybridized to multiple bands of B. henselae genomic DNA digested with several different restriction endonucleases. Thus, the gene is present in multiple copies on the genome or in different arrangements within a given population of B. henselae cells. The gene coding for PapA has been sequenced and codes for a 326-amino-acid protein with a deduced molecular weight of 36,161 daltons. The deduced protein shows 33.3% identity over a 108-amino-acid sequence with the P-min gene product of Escherichia coli. P-min is partially located within the invertible P region of the excisable element e14, found on the E. coli chromosome. Taken together, these results suggest that papA is present on a mobile genetic element of the B. henselae genome and is also packaged into the bacteriophage particle.

Bacteriophage-like particle of Rochalimaea henselae.

An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae. This particle has at least three associated proteins and contains 14 kbp linear DNA segments that are heterogeneous in sequence. The 14 kbp DNA was also present in R. henselae cells as an extrachromosomal element for all 14 strains tested. Despite attempts to induce lysis of R. henselae, plaque formation was not observed. A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B. bacilliformis.

Growth characteristics of Bartonella henselae in a novel liquid medium: primary isolation, growth-phase-dependent phage induction, and metabolic studies.

Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 x 10(8) CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.

The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae.

We present the complete genomes of two human pathogens, Bartonella quintana (1,581,384 bp) and Bartonella henselae (1,931,047 bp). The two pathogens maintain several similarities in being transmitted by insect vectors, using mammalian reservoirs, infecting similar cell types (endothelial cells and erythrocytes) and causing vasculoproliferative changes in immunocompromised hosts. A primary difference between the two pathogens is their reservoir ecology. Whereas B. quintana is a specialist, using only the human as a reservoir, B. henselae is more promiscuous and is frequently isolated from both cats and humans. Genome comparison elucidated a high degree of overall similarity with major differences being B. henselae specific genomic islands coding for filamentous hemagglutinin, and evidence of extensive genome reduction in B. quintana, reminiscent of that found in Rickettsia prowazekii. Both genomes are reduced versions of chromosome I from the highly related pathogen Brucella melitensis. Flanked by two rRNA operons is a segment with similarity to genes located on chromosome II of B. melitensis, suggesting that it was acquired by integration of megareplicon DNA in a common ancestor of the two Bartonella species. Comparisons of the vector-host ecology of these organisms suggest that the utilization of host-restricted vectors is associated with accelerated rates of genome degradation and may explain why human pathogens transmitted by specialist vectors are outnumbered by zoonotic agents, which use vectors of broad host ranges.